Cytochrome P450 monooxygenases

ABSTRACT

Cytochrome P450 II  dependent monooxygenases and DNA molecules encoding these monooxygenases are provided, which are able to catalyze the biosynthethic conversion of aldoximes to nitrils and the conversion of said nitrils to the corresponding cyanohydrins, which are the presursors of cyanogenic glycosides. Moreover, the invention provides methods for obtaining DNA molecules according to the invention and methods for obtaining transgenic plants resistant to insects, acarids, or nematodes or plants with improved nutritive value.

[0001] The present invention relates to genetic engineering in plantsusing recombinant DNA technology in general and to enzymes involved inthe biosynthesis of cyanogenic glycosides and genes encoding theseenzymes in particular. The proteins and genes according to the inventioncan be used to improve the nutritive value or pest resistance of plants.

[0002] Cyanogenic glycosides constitute secondary plant metabolites inmore than 2000 plant species. In some instances they are the source ofHCN which can render a plant toxic if it is taken as food. For examplethe tubers of the cyanogenic crop cassava (Manihot esculenta) constitutean important staple food in tropical areas. The cyanogenic glycosidespresent in the tubers may cause cyanide poisoning in humans due toinsufficiently processed cassava products. Other plant species whoseenzymatic production of HCN accounts for their potential toxicity iftaken in excess as food or used as animal feed include white clover(Trifolium repens), sorghum (Sorghum bicolor), linen flax (Linumusitatissimum), triglochinin (Triglochin maritima), lima beans(Phaseolus lunatus), almonds (Amygdalus) and seeds of apricot (Prunus),cherries and apple (Malus). The toxic properties could be reduced byblocking the biosynthesis of cyanogenic glycosides in these plants.

[0003] The primary precursors of the naturally occuring cyanogenicglycosides are restricted to the five hydrophobic protein amino acidsvaline, leucine, isoleucine, phenylalanine and tyrosine and to a singlenon-protein amino acid, cyclopentenylglycine. These amino acids areconverted in a series of reactions to cyanohydrins which are ultimatelylinked to a sugar residue. Amygdalin for example constitutes theO-β-gentiobioside and prunasin the O-β-glucoside of (R)-mandelonitrile.Another example of cyanogenic glycosides having aromatic aglycones isthe epimeric pair of the cyanogenic glycosides dhurrin and taxiphyllinwhich are to be found in the genus Sorghum and Taxus, respectively.p-Hydroxymandelonitrile for example is converted into dhurrin(β-D-glucopyranosyloxy-(S)-p-hydroxymandelonitrile) by aUDPG-glycosyltransferase. Similiar glycosyltransferases are believed tobe present in most plants. Vicianin and lucumin are further examples fordisaccharide derivatives similiar to amygdalin. Sambunigrin contains(S)-mandelonitrile as its aglycone and is therefore epimeric toprunasin. Examples of cyanogenic glycosides having aliphatic aglyconesare linamarin and lotaustralin found in clover, linen flax, cassava andbeans. A detailed review on cyanogenic glycosides and their biosynthesiscan be found in Conn, Naturwissenschaften 66:28-34, 1979, hereinincorporated by reference.

[0004] The biosynthetic pathway for the cyanogenic glucoside dhurrinderived from tyrosine has been extensively studied (Halkier et al,‘Cyanogenic glucosides: the biosynthetic pathway and the enzyme systeminvolved’ in: ‘Cyanide compounds in biology’, Wiley Chichester (CibaFoundation Symposium 140), pages 49-66, 1988; Halkier and Moller, PlantPhysiol. 90:1552-1559, 1989; Halkier et al, The J. of Biol. Chem.264:19487-19494, 1989; Halkier and Moller, Plant Physiol. 96:10-17,1990, Halkier and Moller, The J. of Biol. Chem. 265:21114-21121, 1990;Halkier et al, Proc. Natl. Acad. Sci. USA 88:487-491, 1991; Sibbesen etal, in: ‘Biochemistry and Biophysics of cytochrome P450. Structure andFunction, Biotechnological and Ecological Aspects’, Archakov, A. I.(ed.), 1991, Koch et al, 8th Int. Conf. on Cytochrome P450, AbstractPII.053; and Sibbesen et al, 8th Int. Conf. on Cytochrome P450, AbstractPII.016). L-Tyrosine is converted to p-hydroxy-mandelonitrile (theprecursor of dhurrin), with N-hydroxytyrosine, N,N-dihydroxytyrosine,(E)- and (Z)-p-hydroxyphenylacetaldehyd oxime, andp-hydroxyphenylacetonitrile being intermediates. Two monooxygenases ofthe cytochrome P450 type are involved in this pathway. In cassava asimiliar pathway involving cytochrome P450 dependent monooxygenases isused for the synthesis of linamarin and lotaustralin from valine andisoleucine, respectively (Koch et al, Archives of Biochemistry andBiophysics, 292:141-150, 1992). The complex pathway from L-tyrosine top-hydroxy-mandelonitrile in Sorghum bicolor was demonstrated to requiretwo multi-functional cytochrome P450 dependent monooxygenases only. Thefirst enzyme, designated P450_(TYR), converts tyrosine top-hydroxyphenylacetaldehyd oxime. The second enzyme, designatedP450_(OX), converts the aldoxime to p-hydroxy-mandelonitrile. In view ofthe similiarities between the biosynthetic pathways of cyanogenicglucosides in different plants it is generally assumed that saidpathways involve two multifunctional P450 dependent monooxygenases,P450_(I) and P450_(II), which convert the precursor amino acid to thecorresponding aldoxime and the aldoxime to the correspondingcyanohydrin, respectively. P450_(I) is a specific enzyme whichdetermines the substrate specificity and, thus, the type of glucosideproduced, whereas P450_(II) is expected to be less specific inconverting a range of structurally different aldoximes into thecorresponding cyanohydrin. Glucosinolates are hydrophilic, non-volatilethioglycosides found within several orders of dicotyledoneousangiosperms (Cronquist, ‘The Evolution and Classification of FloweringPlants, New York Botanical Garden, Bronx, 1988). The occurance ofcyanogenic glucosinolates and glucosides is mutually exclusive. Thegreatest economic significance of glucosinolates is their presence inall members of the Brassicaceae (order of Capparales), whose manycultivars have for centuries provided mankind with a source ofcondiments, relishes, salad crops and vegetables as well as fodders andforage crops. More recently, rape (especially Brassica napus andBrassica campestris) has emerged as a major oil seed of commerce. About100 different glucosinolates are known possessing the same generalstructure but differing in the nature of the side chain. Glucosinolatesare formed from protein amino acids either directly or after a single ormultiple chain extension (Underhill et al, Biochem. Soc. Symp.38:303-326, 1973). N-hydroxy amino acids and aldoximes which have beenidentified as intermediates in the biosynthesis of cyanogenic glycosidesalso serve as efficient precursors for the biosynthesis ofglucosinolates (Kindl et al, Phytochemistry 7:745-756, 1968; Matsuo etal, Phytochemistry 11:697-701, 1972; Underhill, Eur. J. Biochem.2:61-63, 1967). Cytochrome P450_(I) involved in cyanogenic glycosidesynthesis is thus functionally very similiar to the correspondingbiosynthetic enzyme in glucosinolate synthesis, and is thereforeexpected to be a member of the same family of P450 enzymes. Thus we haveisolated a cDNA clone from Sinapis alba encoding a P450 enzyme (SEQ IDNO:17) with 54% identity to P450_(TYR) (CYP79) and catalyzing the firststep in the biosynthesis of glucosinolates, that is the formation of thealdoxime from the parent amino acid. This cDNA clone shows approximately90% identity to an Aribidopsis EST sequence (T42902) which stronglyindicates that this cytochrome P450 enzyme is highly conserved inglucosinolate containing species.

[0005] The reduction of the complex biosynthetic pathway forcyanohydrins described above to the catalytic activity of only twoenzymes, cytochrome P450_(I) and P450_(II), allows for the manipulationof the biosynthetic pathway of cyanogenic glucosides in plants. Bytransfection of gene constructs coding for one or both of themonooxygenases a biosynthetic pathway for cyanogenic glucosides caneither be modified, reconstituted, or newly established.

[0006] The modification or introduction of a biosynthetic pathway forcyanogenic glycosides in plants by methods known in the art is of greatinterest, since cyanogenic glycosides can be toxic to insects, acarids,and nematodes. Therefore, the modification, introduction orreconstitution of a biosynthetic pathway for cyanogenic glycosides inplants or certain plant tissues will allow to render plants unpalatablefor insects, acarids or nematodes and thus help to reduce the damage tothe crop by pests. In combination with other insecticidal principlessuch as Bacillus thuringiensis endotoxins the damage to the crop bypests could be even further reduced.

[0007] Alternatively, the sequences of the genes encoding themonooxygenases according to the invention can be used to design DNAplasmids which upon transfection into a plant containing cyanogenicglycosides such as cassava, sorghum or barley, eliminate cyanogenicglycosides normally produced in wildtype plants. This can be achieved byexpression of antisense or sense RNA or of ribozymes as described inEP-458367-A1, EP-240208-A2, U.S. Pat. No. 5,231,020, WO89/05852, andWO90/11682 which inhibits the expression of monooxygenases according tothe invention. This is of great interest as in spite of numerous effortsit has not been possible through traditional plant breeding tocompletely remove the cyanogenic glycosides from for example cassava orsorghum. On the other hand it has been shown that elevated amounts ofcyanogenic glycosides in the epidermal cells of barley cultivars conferincreased sensitivity to attack by the mildew fungus Erysiphe graminis(Pourmohensi, PhD thesis, Göttingen, 1989; Ibenthal et al, Angew. Bot.67:97-106, 1993). A similiar effect has been observed in the cyanogenicrubber tree Hevea brasiliensis upon attack by the fungus Microcyclusulei (Lieberei et al, Plant Phys. 90:3-36, 1989) and with flax attackedby Colletotrichum lini (Lüdtke et al, Biochem. Z. 324:433-442, 1953). Inthese instances the quantitative resistance of the plants stipulatedabove and of other plants, where cyanogenic glycosides confer increasedsensitivity to attack by microorganisms, can be increased by preventingthe production of cyanogenic glycosides in such plants. In barley, thecyanogenic glycosides are located in the epidermal cells. The expressionof antisene, sense or ribozyme constructs is therefore preferably butnot necessarily driven by an epidermis specific promoter.

[0008] The presence of even minor amounts of cyanogenic glycosides inplants may also cause nutritional problems due to generation of unwantedcarcinogens as demonstrated in barley. Barley malt for example containslow amounts of the cyanogenic glucoside epiheterodendrin which in thecause of production of grain-based spirits can be converted toethylcarbamate which is considered to be a carcinogen. Attempts arebeing made to introduce mandatory maximum allowable concentrations ofethylcarbamate in fermented food, beverages and spirits (Food ChemicalNews 29:33.35, 1988).

[0009] WO 95/16041 describes a DNA molecule coding for a cytochromeP450_(I) monooxygenase, which catalyzes the conversion of an amino acidto the corresponding N-hydroxyamino acid, N,N-dihydroxyamino acid, andthe conversion of the N,N-dihydroxyamino acid to the correspondingaldoxime. The parent amino acid is selected from the group consisting oftyrosine, phenylalanine, tryptophan, valine, leucine, isoleucine andcyclopentenylglycine. The DNA molecules either correspond to naturallyoccuring genes or to functional homologues thereof which are the resultof mutation, deletion, truncation, etc. but still encode a cytochromeP450_(I) monooxygenase capable of catalyzing more than one reaction ofthe biosynthetic pathway of cyanogenic glycosides. The monooxygenasespreferably contain a single catalytic center.

[0010] Additionally WO 95/16041 describes DNA molecules coding forcytochrome P450_(II) monooxygenases such as P450_(OX) of Sorghum bicolor(L.) Moench. They catalyze the conversion of an aldoxime to a nitrileand the conversion of the nitrile to the corresponding cyanohydrin. Thecatalysis of the conversion of tyrosine into p-hydroxyphenylacetonitrileby two multifunctional P450 enzymes explains why all intermediates inthis conversion except (Z)-p-hydroxyphenylacetaldoxime are channelled.The strategy suggested for the isolation of P450_(OX) is based on thatused for the isolation of P450_(TYR) (CYP79, Sibbesen et al, Proc. Natl.Acad Sci. USA 91: 9740-9744, 1994) from sorghum. In this approach a DEAESepharose ion exchange column serves to bind P450 enzymes whereas theyellow pigments in the sample do not bind. Removal of the pigmentsserves a dual purpose. It is a prerequisite for binding of P450 enzymsto the subsequent columns, and it enables assessment of the content ofP450 by spectrometry (carbon monoxide and substrate binding). Thepresent invention demonstrates that P450_(OX) in contrast shows a lowbinding affinity to the DEAE column and is essentially recovered in therun through and wash fractions. To separate P450_(OX) activity from theyellow pigments by a Triton X-114 based phase partitioning procedure isapplied. Using preferentially 0.6 to 1% Triton X-114, P450_(OX) is foundto partition to both phases in contrast to P450_(TYR), which isrecovered in the detergent rich upper phase. By increasing theconcentration of Triton X-114 up to 6%, the majority of P450_(OX) isrecovered from the detergent poor lower phase, while the yellow pigmentsare present in the upper phase. A disadvantage of using 6% Triton X-114is an enhancement of the conversion of P450_(OX) into its denatured P420form. This knowledge is used in the present invention to purify for thefirst time P450_(II) monooxygenases such as P450_(OX), to clone thegenes encoding the monooxygenases, and to stably transform plants withthe monooxygenase encoding genes. The isolation of P450_(OX) anddetermination of partial amino acid sequences permit the design ofoligonucleotide probes and the isolation of a cDNA encoding P450_(OX).However, in the present case cloning was accomplished via an independentapproach.

[0011] The invention relates primarily to DNA molecules encodingcytochrome P450_(II) monooxygenases, which catalyze the conversion of analdoxime to a nitrile and the conversion of said nitrile to thecorresponding cyanohydrin. Preferably the aldoxime is the product of aconversion of an amino acid selected from the group consisting oftyrosine, phenylalanine, tryptophan, valine, leucine, isoleucine andcyclopentenylglycine or an amino acid selected from the group consistingof L-tyrosine, L-valine and L-isoleucine, catalyzed by a P450_(I)monooxygenase as described in WO 95/16041. The DNA molecules accordingto the invention either correspond to naturally occuring genes or tohomologues thereof which are the result of mutation, deletion,truncation, etc. but still encode a cytochrome P450_(II) monooxygenase,which catalyzes the conversion of an aldoxime to a nitrile and thesubsequent conversion of said nitrile to the corresponding cyanohydrin.The monooxygenases according to the invention catalyze more than onereaction of the biosynthetic pathway of cyanogenic glycosides andpreferably contain a single catalytic center.

[0012] Cytochrome P450_(II) enzymes might be present in most livingorganisms. The DNA molecules according to the present invention encodingP450_(II) monooxygenases are structurally and functionally similar toDNA molecules obtainable from various plants which produce cyanogenicglycosides. In a preferred embodiment of the invention the DNA moleculeshybridize to a fragment of the DNA molecule with the nucleotide sequencegiven in SEQ ID NO:1. Said fragment is more than 10 nucleotides long andpreferably longer than 15, 20, 25, 30, or 50 nucleotides. Factors thataffect the stability of hybrids determine the stringency ofhybridization conditions and can be measured in dependence of themelting temperature T_(m) of the hybrids formed. The calculation ofT_(m) is desribed in several textbooks. For example Keller et aldescribe in: “DNA Probes: Background, Applications, Procedures”,Macmillan Publishers Ltd, 1993, on pages 8 to 10 the factors to beconsidered in the calculation of T_(m) values for hybridizationreactions. The DNA molecules according to the present inventionhybridize with a fragment of SEQ ID NO:1 at a temperatur 30° C. belowthe calculated T_(m) of the hybrid to be formed. Preferably theyhybridize at temperatures 25, 20, 15, 10, or 5° C. below the calculatedT_(m).

[0013] For the purposes of gene manipulation using recombinant DNAtechnology the DNA molecule according to the invention may in additionto the gene coding for the monooxygenase comprise DNA which allows forexample replication and selection of the inventive DNA in microorganismssuch as E. coli, Bacillus, Agrobacterium, Streptomyces or yeast. It mayalso comprise DNA which allows the monooxygenase genes to be expressedand selected in homologous or heterologous plants. Such sequencescomprise but are not limited to genes the codon usage of which has beenadapted to the codon usage of the heterologous plant as described inWO93/07278; to genes conferring resistance to neomycin, kanamycin,methotrexate, hygromycin, bleomycin, streptomycin, or gentamycin, toaminoethylcystein, glyophosphate, sulfonylurea, or phosphinotricin; toscorable marker genes such as galactosidase; to its natural promoter andtranscription termination signals; to promoter elements such as the 35Sand 19S CaMV promoters, or tissue specific plant promoters such aspromoters specific for root (described for example in EP-452269-A2,WO91/13992, U.S. Pat. No. 5,023,179), green leaves such as the maizephosphoenol pyruvate carboxylase (PEPC), pith or pollen (described forexample in WO93/07278), or inducible plant promoters (EP-332104); and toheterologous transcription termination signals.

[0014] The present invention also relates to the P450_(II)monooxygenases which catalyze the conversion of an aldoxime to a nitrileand the conversion of said nitrile to the corresponding cyanohydrine. Ina preferred embodiment of the invention the monooxygenases are purifiedand can be used to establish monoclonal or polyclonal antibodies whichspecifically bind to the monooxygenases. In particular cytochromeP450_(OX) having a molecular weight of 55 kD as determined by SDS-PAGEis isolated from Sorghum bicolor (L.) Moench. Its amino acid sequence isgiven in SEQ ID NO:2.

[0015] The catalytic properties of P450_(OX) resembles those of acytochrome P450 activity reported in microsomes from rat liver (DeMasteret al, J. Org. Chem. 5074-5075, 1992). A characteristic of cytochromeP450_(OX) and of other members belonging to the cytochrome P450_(OX)family is that dehydration of the aldoxime to the corresponding nitrileis dependent on the presence of NADPH but that this dependence in somecases can be overcome by the addition of sodium dithionite or otherreductants.

[0016] Of all known sequences for cytochrome P450 enzymes, cytochromeP450_(OX) shows the highest amino acid sequence identity (44%) to theavocado enzyme CYP71A1 and less than 40% identity to all other membersof the CYP71 family. Avocados, do not produce cyanogenic glycosides andCYP71A1 does not catalyze the conversion of an aldoxime to a nitrile andthe conversion of said nitrile to the corresponding cyanohydrin. Thus,according to the present invention a family of cytochrome P450_(II)monooxygenases can be defined the members of which catalyze theconversion of an aldoxime to the corresponding cyanohydrin and have a40% or higher amino acid sequence identity to that of cytochromeP450_(OX). Preferably the amino acid sequence identity with cytochromeP450_(OX) is higher than 50% or higher than 55%. It is suggested toassign P450_(OX) the first member of a new CYP71 subfamily (CYP71E1) asit clusters with other CYP71 sequences in dendrograms, the graphicaloutput of a multiple sequence alignment. Generally, according to thenomenclature commitee, less than 40% sequence identity on the amino acidlevel is required for a cytochrome P450 to be assigned to a new CYPfamily and sequences that are more than 55% identical are assigned tothe same subfamily. When making multiple sequence alignments not onlysequence identities but also sequence similarities such as same netcharge or a comparable hydrophobicity/hydrofilicity of the individualamino acids are considered. In such alignments P450_(OX) clusters withthe other CYP71 sequences and should therefore be included in the CYP71family despite the fact that it shows less than 40% identity to allother members of the CYP71 family except CYP71A1 from avocado. As itshows low sequence identity to the other members it ought to be assignedto a new subfamily. The other CYP71 family members are all fromnon-cyanogenic species and their function is unknown. The catalyticproperties of the previously identified P450s belonging to the CYP71family remain elusive. They are thought to be involved in terpenehydroxylations. None of them has been suggested to utilize oximes assubstrates nor to be multifunctional converting aldoximes into nitrilesand cyanohydrins.

[0017] A further embodiment of the present invention is to be seen in amethod for the preparation of cDNA coding for a cytochrome P450_(II)monooxygenase, which catalyzes the conversion of an aldoxime to anitrile and the conversion of said nitrile to the correspondingcyanohydrin. It comprises

[0018] (a) isolating and solubilizing microsomes from plant tissueproducing cyanogenic glycosides,

[0019] (b) purifying the cytochrome P450 monooxygenase,

[0020] (c) raising antibodies against the purified monooxygenase,

[0021] (d) probing a cDNA expression library of plant tissue producingcyanogenic glycosides with said antibody, and

[0022] (e) isolating clones which express the monooxygenase.

[0023] Microsomes can be isolated from plant tissues which show a highactivity of the enzyme system responsible for biosynthesis of thecyanogenic glycosides. These tissues may be different from plant speciesto plant species. A preferred source of microsomes are freshly isolatedshoots harvested 1 to 20 days, preferably 2 to 10 days and mostpreferably 2 to 4 days after germination. Etiolated seedlings arepreferred from plant producing cyanogenic glycosides but light grownseedlings may also be used. Following isolation the microsomes aresolubilized in buffer containing one or more detergents. Preferreddetergents are RENEX 690 (J. Lorentzen A/S, Kvistgard, Denmark), reducedTriton X-100 (RTX-100), Triton X-114, and CHAPS.

[0024] The cytochrome P450 monooxygenases can be purified applyingstandard techniques for protein purification such asultracentrifugation, fractionated precipitation, dialysis, SDS-PAGE andcolumn chromatography. Possible columns comprise but are not limited toion exchange columns such as DEAE Sepharose, Reactive dye columns suchas Cibacron yellow 3 agarose, Cibacron blue agarose and Reactive red 120agarose, and gel filtration columns such as Sephacryl S-1000. Thecytochrome P450 content of the individual fractions can be determinedfrom carbon monoxide difference spectra. A special difficulty during theisolation of P450_(OX) which also renders quantification of P450_(OX)difficult is its co-migration with yellow pigments during the initialpurification steps instead of binding to the ion exchange columnnormally used for purification of P450 enzymes such as for exampleP450_(TYR). The presence of yellow pigments prevents the binding ofP450_(OX) to a number of different column materials and thus constitutesa major obstacle towards further purification. Separation of P450_(OX)from the yellow pigments could, however, be accomplished by temperatureinduced Triton X-114 phase partitioning. The method was optimized withrespect to P450_(OX) recovery and removal of pigments by increasing theamount of Triton X-114. At 6%, which is six to ten fold the level usedfor other P450s, approximately 80% of the P450_(OX) activity partitionsto the clear lower phase. Little purification besides the removal ofyellow pigments is achieved in this purification step. However, when theP450_(OX) containing lower phase is applied to a Cibacron blue dyecolumn, salt gradient elution produced nearly homogeneous P450_(OX) asjudged from the presence of a major Coomassie stained band with anapparent molecular mass of 55 kDa in those fractions which byreconstitution showed P450_(OX) activity. Isolated P450_(OX) produced acarbon monoxide spectrum with an absorption peak at 450 nm but arelatively large part of the isolated enzyme was present in thedenatured P420 form. Quantitative determination of the total content andspecific activity of P450_(OX) at the different steps in the isolationprocedure was hampered by the continuous conversion of P450_(OX) intothe denatured P420 form. In addition, the specific activity of P450_(OX)is dependent on the inhibitory effects exerted by the differentdetergents used. The total P450 content of the fractions is thus to beconsidered semiquantitative.

[0025] The purified proteins can be used to elicit antibodies in forexample mice, goats, sheeps, rabbits or chickens upon injection. 5 to 50μg of protein are injected several times during approximately 14 dayintervals. In a preferred embodiment of the invention 10 to 20 μg areinjected 2 to 6 times in 14 day intervals. Injections can be done in thepresence or absence of adjuvants. Immunoglobulins are purified from theantisera and spleens can be used for hybridoma fusion as described inHarlow and Lane, ‘Antibodies: A Laboratory Manual’, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1988, herein incorporated byreference. Antibodies specifically binding to a cytochrome P450_(II)monooxygenase can also be used in plant breeding to detect plantsproducing altered amounts of cytochrome P450 monooxygenases and thusaltered amounts of cyanogenic glycosides.

[0026] The methods for the preparation of plant tissue cDNA librariesare extensively described in Sambrook et al, Molecular cloning: Alaboratory manual. Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989, the essential parts of which regarding preparationof cDNA libraries are herein incorporated by reference. PolyA⁺ RNA isisolated from plant tissue which shows a high activity of the enzymesystem responsible for biosynthesis of the cyanogenic glycosides. Thesetissues may be different from plant species to plant species. Apreferred tissue for polyA⁺ RNA isolation is the tissue of freshlyisolated shoots harvested 1 to 20 days, preferably 2 to 10 days and mostpreferably 2 to 4 days after germination. The obtained cDNA librariescan be probed with antibodies specifically binding the cytochromeP450_(II) monooxygenase and clones expressing the monooxygenase can beisolated.

[0027] An alternative method for the preparation of cDNA coding for acytochrome P450_(II) monooxygenase comprises

[0028] (a) isolating and solubilizing microsomes from plant tissueproducing cyanogenic glycosides,

[0029] (b) purifying the cytochrome P450_(II) monooxygenase,

[0030] (c) obtaining a complete or partial protein sequence of themonoxygenase,

[0031] (d) designing oligonucleotides specifying DNA coding for 4 to 15amino acids of said monooxygenase protein sequence

[0032] (e) probing a cDNA library of plant tissue producing cyanogenicglycosides with said oligonucleotides, or DNA molecules obtained fromPCR amplification of cDNA using said oligonucleotides, and

[0033] (f) isolating clones which encode cytochrome P450_(II)monooxygenase.

[0034] Amino acid sequences of internal peptides which are the result ofprotease digestion can be obtained by standard techniques such as Edmandegradation. Oligonucleotides specifying DNA coding for partial proteinsequences of the inventive monooxygenases are obtained by reversetranslation of parts of the protein sequence according to the geneticcode. Protein sequences encoded by DNA sequences of low degeneracy arepreferred for reverse translation. Their length ranges from 4 to 15 andpreferably from 5 to 10 amino acids. If necessary the codons used in theoligonucleotides can be adapted to the codon usage of the plant source(Murray et al, Nucleic Acids Research 17:477-498, 1989). The obtainedoligonucleotides can be used to probe cDNA libraries as described inSambrook et al, (Molecular cloning: A laboratory manual. Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) for cloneswhich are able to basepair with said oligonucleotides. Alternatively,oligonucleotides can be used in a polymerase chain reaction, themethodology of which is known in the art, with plant cDNA as thetemplate for amplification. In this case the obtained amplificationproducts are used to probe the cDNA libraries. Clones encodingcytochrome P450_(II) monooxygenases are isolated.

[0035] An alternative method of cloning genes is based on theconstruction of a gene library composed of expression vectors. In thatmethod, analogously to the methods already described above, genomic DNA,but preferably cDNA, is first isolated from a cell or a tissue capableof expressing a P450_(II) monooxygenase and is then spliced into asuitable expression vector. The gene libraries so produced can then bescreened using suitable means, preferably antibodies. Clones whichcomprise the desired gene or at least part of the gene as an insert areselected.

[0036] Alternatively, cDNA molecules coding for a cytochrome P450monooxygenase which catalyzes conversion of an aldoxime to a nitrile andconversion of said nitrile to the corresponding cyanohydrin; can beachieved by

[0037] (a) designing degenerated oligonucleotides covering 3 to 10 aminoacids of conserved regions of A-type cytochromes,

[0038] (b) using the degenerated oligonucleotides to amplify one or morecytochrome specific DNA fragments using the polymerase chain reaction,

[0039] (c) screening a cDNA library with the cytochrome specificfragments to obtain full length cDNA,

[0040] (d) expressing the full length cDNA in a microbial host,

[0041] (e) identifying hosts expressing cytochrome P450 monooxygenasewhich catalyzes the conversion of an aldoxime to a nitrile and theconversion of said nitrile to the corresponding cyanohydrin, and

[0042] (f) purifying the cloned DNA from said host.

[0043] Total DNA from a DNA library, preferably from a cDNA library, canbe used as template in a PCR reaction with one or more primersrepresenting conserved regions of A-type cytochromes (Durst et al, DrugMetabolism and Drug Interactions 12: 189-206, 1995) which are believedto be derived from a common plant cytochrome P450 ancestor. Based on amultiple sequence alignment of A-type cytochromes P450 three highlyconserved regions on the amino acid level can be defined: region 1(V/I)KEX(L/F)R, region 2 FXPERF, and region 3 PFGXGRRXCXG. Degenerateinosine (I) containing primers can be designed each covering 3 to 10 andpreferably about 5 or 6 amino acids of the two regions respectively. PCRis for example performed in three consecutive rounds. Round 1 using aprimer covering the consensus region FXPERF and a standard T7 primercovering the T7 promoter in the library vector amplifies cDNAs derivedfrom mRNAs encoding A-type cytochromes P450. A second round of PCR usingprimers covering the two consensus regions and the amplified DNA ofround 1 as template preferentially amplifies a 100 bp fragment which isthen ligated into pBluescript and sequenced. Gene specific primers aredesigned based on the DNA sequence obtained. They are used in round 3 incombination with a primer complementary to the poly A tail (primer dT+V)and DNA of PCR round 1 as the template to amplify an approximately 500bp DNA fragment which can be used as a gene specific probe to isolatefull-length cDNAs. This PCR approach is not unique to the isolation ofP450_(OX) but is general for the isolation of A-type cytochromes P450.The A-type cytochromes P450 obtained need to be heterologously expressedto determine their function.

[0044] cDNA clones or PCR products prepared as described above orfragments thereof may be used as a hybridization probe in a process ofidentifying further DNA sequences encoding a protein product thatexhibits P450_(II) monooxygenase activity from a homologous or aheterologous source organism such as fungi or heterologous plants. Asuitable source is tissue from plants containing cyanogenic glycosides.Said clones or PCR products may also be used as an RFLP marker todetermine, for example, the location of the cytochrome P450monooxygenase gene or a closely linked trait in the plant genome or formarker assisted breeding [EP-A 306139; WO 89/07647].

[0045] Using the methods described above it is possible to isolatevarious genes that code for a P450_(II) monooxygenase. Said genes can beused in a method for producing a purified recombinant cytochromeP450_(II) monooxygenase which catalyzes the conversion of an aldoxime toa nitrile and the conversion of said nitrile to the correspondingcyanohydrin; comprising

[0046] (a) engineering the gene encoding said monooxygenase to beexpressible in a host organism such as bacteria, yeast or insect cells,

[0047] (b) transforming said host organism with the engineered gene, and

[0048] (c) isolating the protein from the host organism or the culturesupernatant.

[0049] In a preferred embodiment of the invention the method is used toobtain purified recombinant cytochrome P450_(OX), or cytochromeP450_(OX) which has been modified by known techniques of genetechnology. Preferably the modifications lead to increased expression ofthe recombinant protein or to altered substrate specificity.

[0050] The inventive DNA molecules can be used to obtain transgenicplants resistant to insects or acarids. Specific embodiments are listedbut not limited to those in Table B of WO 95/16041 (page 45) as well asto nematodes described below. For convenience only said Table is notrepeated in this specification but it is meant to be incorporated hereinby referring to the disclosure of WO 95/16041. Preferably the transgenicplants are resistant to Coleoptera and Lepidoptera such as western cornroot worm (Diabrotica virgifera virgifera), northern corn root worm(Diabrotica longicornis barberi), southern corn rootworm (Diabroticaundecimpunctata howardi), cotton bollworm, European corn borer, cornroot webworm, pink bollworm and tobacco budworm. Nematodes are theprincipal animal parasites of plants causing global losses toagriculture estimated at >$100 billion each year. Certain nematodesinduce feeding sites involving plant cell modification and feeding atone site for several hours or considerably more. They include species ofthe genera Meloidogyne Globodera, Heterodera, Rotylenchulus,Tylenchulus, Naccobus, Xiphinema, Longidorus, Paralongidorus,Cryphodera, Trophotylenchulus, Hemicycliophora, Criconemella, Verutusand Heliocotylenchus. Genera considered to feed for a more restrictedperiod at one site include Pratylenchus, Radopholus, Hirschmanniella,Trichodorus, Paratrichodorus, Ditylenchus, Aphelenchoides, Scutellonema,and Belonolaimus.

[0051] The transgenic plants comprise DNA coding for the newmonooxygenases which catalyze the conversion of said aldoxime to anitrile and the conversion of said nitrile to the correspondingcyanohydrine. In addition the transgenic plants may comprisemonooxygenase genes genetically linked to herbicide resistance genes.The transgenic plants are preferably monocotyledoneous ordicotyledoneous plants. Specific embodiments are listed in Table A of WO95/16041 (pages 33-44). For convenience only said Table is not repeatedin this specification but it is meant to be incorporated herein byreferring to the disclosure of WO 95/16041. Preferably they are selectedfrom the group consisting of maize, rice, wheat, barley, sorghum,cotton, soybeans, sunflower, grasses, oil seed rape, sugar beet,broccoli, cauliflower, cabbage, cucumber, sweet corn, daikon, benas,lettuce, melon, pepper, squash, tomato, and watermelon. The plants canbe obtained by a method comprising

[0052] (a) introducing into a plant cell or plant tissue which can beregenerated to a complete plant, DNA comprising a gene expressible inthat plant encoding an inventive monooxygenase, and

[0053] (b) selecting transgenic plants.

[0054] Similarly the inventive DNA molecules can be used to obtaintransgenic plants expressing anti-sense or sense RNA or ribozymestargeted to the genes of the endogenous P450_(II) monooxygenases.Expression of these molecules in transgenic plants reduces theexpression of cytochrome P450_(II) monooxygenases. Such plants showimproved disease resistance or nutritive value due to reduced expressionof cyanogenic glycosides. The plants can be obtained with a methodcomprising

[0055] (a) introducing into a plant cell or tissue which can beregenerated to a complete plant, DNA encoding sense RNA, anti sense RNAor a ribozyme, the expression of which reduces the expression ofcytochrome P450_(II) monooxygenases, and

[0056] (b) selecting transgenic plants.

[0057] A number of very efficient processes are available forintroducing DNA into plant cells, which processes are based on the useof gene transfer vectors or on direct gene transfer processes.

[0058] One possible method of inserting a gene construct into a cellmakes use of the infection of the plant cell with Agrobacteriumtumefaciens and/or Agrobacterium rhizogenes, which has been transformedwith the said gene construction. The transgenic plant cells are thencultured under suitable culture conditions known to the person skilledin the art, so that they form shoots and roots and whole plants arefinally formed.

[0059] Within the scope of this invention is the so-called leaf disktransformation using Agrobacterium (Horsch et al, Science 227:1229-1231,1985). Sterile leaf disks from a suitable target plant are incubatedwith Agrobacterium cells comprising one of the chimaeric geneconstructions according to the invention, and are then transferred intoor onto a suitable nutrient medium. Especially suitable, and thereforepreferred within the scope of this invention, are LS media that havebeen solidified by the addition of agar and enriched with one or more ofthe plant growth regulators customarily used, especially those selectedfrom the group of the auxins consisting of α-naphthylacetic acid,picloram, 2,4,5-trichlorophenoxyacetic acid, 2,4-dichlorophenoxyaceticacid, indole-3-butyric acid, indole-3-lactic acid, indole-3-succinicacid, indole-3-acetic acid and p-chlorophenoxyacetic acid, and from thegroup of the cytokinins consisting of kinetin, 6-benzyladenine,2-isopentenyladenine and zeatin. The preferred concentration of auxinsand cytokinins is in the range of 0.1 mg/l to 10 mg/l.

[0060] After incubation for several days, but preferably afterincubation for 2 to 3 days at a temperature of 20° C. to 40° C.,preferably from 23° C. to 35° C. and more preferably at 25° C. and indiffuse light, the leaf disks are transferred to a suitable medium forthe purpose of shoot induction. Especially preferred for the selectionof the transformants is an LS medium that does not contain auxin butcontains cytokinin instead, and to which a selective substance has beenadded. The cultures are kept in the light and are transferred to freshmedium at suitable intervals, but preferably at intervals of one week.Developing green shoots are cut out and cultured further in a mediumthat induces the shoots to form roots. Especially preferred within thescope of this invention is an LS medium that does not contain auxin orcytokinin but to which a selective substance has been added for theselection of the transformants.

[0061] In addition to Agrobacterium-mediated transformation, within thescope of this invention it is possible to use direct transformationmethods for the insertion of the gene constructions according to theinvention into plant material.

[0062] For example, the genetic material contained in a vector can beinserted directly into a plant cell, for example using purely physicalprocedures, for example by microinjection using finely drawnmicropipettes (Neuhaus et al, Theoretical and Applied Genetics74:363-373, 1987), electroporation (D'Halluin et al, The Plant Cell4:1495-1505, 1992; WO92/09696), or preferably by bombarding the cellswith microprojectiles that are coated with the transforming DNA(“Microprojectile Bombardment”; Wang et al, Plant Molecular Biology11:433-439, 1988; Gordon-Kamm et al, The Plant Cell 2:603-618, 1990;McCabe et al, Bio/Technology 11:596-598, 1993; Christou et, PlantPhysiol. 87:671-674, 1988; Koziel et al, Biotechnology 11: 194-200,1993). Moreover, the plant material to be transformed can optionally bepretreated with an osmotically active substance such as sucrose,sorbitol, polyethylene glycol, glucose or mannitol.

[0063] Other possible methods for the direct transfer of geneticmaterial into a plant cell comprise the treatment of protoplasts usingprocedures that modify the plasma membrane, for example polyethyleneglycol treatment, heat shock treatment or electroporation, or acombination of those procedures (Shillito et al, Biotechnology3:1099-1103,1985).

[0064] A further method for the direct introduction of genetic materialinto plant cells, which is based on purely chemical procedures and whichenables the transformation to be carried out very efficiently andrapidly, is described in Negrutiu et al, Plant Molecular Biology8:363-373, 1987.

[0065] Also suitable for the transformation of plant material is directgene transfer using co-transformation (Schocher et al, Bio/Technology4:1093-1096,1986).

[0066] The list of possible transformation methods given above by way ofexample does not claim to be complete and is not intended to limit thesubject of the invention in any way.

[0067] The genetic properties engineered into the transgenic seeds andplants described above are passed on by sexual reproduction orvegetative growth and can thus be maintained and propagated in progenyplants. Generally said maintenance and propagation make use of knownagricultural methods developed to fit specific purposes such as tilling,sowing or harvesting. Specialized processes such as hydroponics orgreenhouse technologies can also be applied. As the growing crop isvulnerable to attack and damages caused by insects or infections as wellas to competition by weed plants, measures are undertaken to controlweeds, plant diseases, insects, nematodes, and other adverse conditionsto improve yield. These include mechanical measures such a tillage ofthe soil or removal of weeds and infected plants, as well as theapplication of agrochemicals such as herbicides, fungicides,gametocides, nematicides, growth regulants, ripening agents andinsecticides.

[0068] Use of the advantageous genetic properties of the transgenicplants and seeds according to the invention can further be made in plantbreeding which aims at the development of plants with improvedproperties such as tolerance of pests, herbicides, or stress, improvednutritional value, increased yield, or improved structure causing lessloss from lodging or shattering. The various breeding steps arecharacterized by well-defined human intervention such as selecting thelines to be crossed, directing pollination of the parental lines, orselecting appropriate progeny plants. Depending on the desiredproperties different breeding measures are taken. The relevanttechniques are well known in the art and include but are not limited tohybridization, inbreeding, backcross breeding, multiline breeding,variety blend, interspecific hybridization, aneuploid techniques, etc.Hybridization techniques also include the sterilization of plants toyield male or female sterile plants by mechanical, chemical orbiochemical means. Cross pollination of a male sterile plant with pollenof a different line assures that the genome of the male sterile butfemale fertile plant will uniformly obtain properties of both parentallines. Thus, the transgenic seeds and plants according to the inventioncan be used for the breeding of improved plant lines which for exampleincrease the effectiveness of conventional methods such as herbicide orpestidice treatment or allow to dispense with said methods due to theirmodified genetic properties. Alternatively new crops with improvedstress tolerance can be obtained which, due to their optimized genetic“equipment”, yield harvested product of better quality than productswhich were not able to tolerate comparable adverse developmentalconditions.

[0069] In seeds production germination quality and uniformity of seedsare essential product characteristics, whereas germination quality anduniformity of seeds harvested and sold by the farmer is not important.As it is difficult to keep a crop free from other crop and weed seeds,to control seedborne diseases, and to produce seed with goodgermination, fairly extensive and well-defined seed production practiceshave been developed by seed producers, who are experienced in the art ofgrowing, conditioning and marketing of pure seed. Thus, it is commonpractice for the farmer to buy certified seed meeting specific qualitystandards instead of using seed harvested from his own crop. Propagationmaterial to be used as seeds is customarily treated with a protectantcoating comprising herbicides, insecticides, fungicides, bactericides,nematicides, molluscicides or mixtures thereof. Customarily usedprotectant coatings comprise compounds such as captan, carboxin, thiram(TMTD®), methalaxyl (Apron®), and pirimiphosmethyl (Actellic®). Ifdesired these compounds are formulated together with further carriers,surfactants or application-promoting adjuvants customarily employed inthe art of formulation to provide protection against damage caused bybacterial, fungal or animal pests. The protectant coatings may beapplied by impregnating propagation material with a liquid formulationor by coating with a combined wet or dry formulation. Other methods ofapplication are also possible such as treatment directed at the buds orthe fruit.

[0070] It is a further aspect of the present invention to provide newagricultural methods such as the methods examplified above which arecharacterized by the use of transgenic plants, transgenic plantmaterial, or transgenic seed according to the present invention.

[0071] The following examples further describe the materials and methodsused in carrying out the invention and the subsequent results. They areoffered by way of illustration, and their recitation should not beconsidered as a limitation of the claimed invention.

EXAMPLES Example 1

[0072] Preparation of Microsomes

[0073] All steps involving the preparation of microsomes are carried outat 4° C. unless otherwise stated. All buffers are degassed by stirringin vacuo and flushed with argon. Seeds of Sorghum bicolor (L.) Moench(hybrid SS1000 from AgriPro, Texas, USA) are germinated in the dark for40 h at 28° C. on metal screens covered with gauze. Microsomes areprepared from approximately 3 cm etiolated seedlings. The seedlings areharvested and homogenized using a mortar and pestle in 2 volumes (v/w)of 250 mM sucrose, 100 mM Tricine (pH 7.9), 2 mM EDTA, and 2 mM DTT.Polyvinylpolypyrrolidone is added (0.1 g/g fresh weight) prior tohomogenization. The homogenate is filtered through 22 μm nylon cloth andcentrifuged for 10 minutes at 16500×g. The supernatant is centrifugedfor 1 hour at 165000×g. The microsomal pellet is resuspended andhomogenized in isolation buffer using a Potter-Elvehjem homogenizerfitted with a teflon pestle. After recentrifugation and rehomogenizationthe homogenate is frozen in liquid nitrogen and stored at −80° C. untiluse.

Example 2

[0074] Enzyme Assays: Determination of Total Cytochrome P450

[0075] Quantitative determination of total cytochrome P450 is carriedout by difference spectroscopy using an extinction coefficient of 91mM⁻¹ cm⁻¹ for the adduct between reduced cytochrome P450 and carbonmonoxide (A₄₅₀₋₄₉₀) (Omura et al, J. Biol. Chem. 239:2370-2378, 1964).

Example 3

[0076] Purification of Cytochrome P450_(OX)

[0077] All steps involving the purification of enzyme are carried out at4° C. unless otherwise stated. Buffer A: Buffer C: 8.9% glycerol 8.9%glycerol 10 mM KH₂PO₄/K₂HPO₄ 40 mM KH₂PO₄/K₂HPO₄ (pH 7.9) (pH 7.9) 0.2mM EDTA 5.0 mM EDTA 2.0 mM DTT 2.0 mM DTT 1.0% (v/v) Renex 690 1.0%(w/v) CHAPS 0.05% RTX-100

[0078] Buffers are degassed three times by stirring in vacuo beforedetergent and DTT are added. Between each degassing the buffer isflushed with argon. The ability of different column fractions tometabolize radiolabeled p-hydroxyphenylacetaldoxime is monitoredthroughout the purification procedure to identify the presence ofP450_(OX) in the fractions.

[0079] Microsomes (400 mg protein in 20 ml) are diluted to 100 ml with abuffer composed of 8.9% glycerol, 10 mM KH₂PO₄/K₂HPO₄ (pH 7.9), 0.2 mMEDTA, 2 mM DTT after which 100 ml of 10 mM KH₂PO₄/K₂HPO₄ (pH 7.9), 8.9%glycerol, 0.2 mM EDTA, 2 mM DTT , 0.1% RTX-100 (v/v), 2% Renex is slowlyadded with constant stirring. After additional stirring for 30 min andsubsequent ultracentrifugation at 150000×g for 35 minutes, theapproximately 190 ml supernatant are applied with a flow rate of 100ml/h to a 5×5 cm column of DEAE Sepharose FF/S-100 Sepharose (20/80 wetvolumes, Pharmacia) equilibrated in buffer A. The DEAE Sepharose ionexchange resin is diluted with S-100 Sepharose gel filtration materialin the ratio 1:4 to avoid too high concentrations of cytochrome P450enzymes upon binding, which could result in irreversible aggregation.The column is then washed with 150 ml buffer A. P450_(OX) binds weaklyto the column and was essentially recovered in those of the run off andwash fractions which contain yellow pigment. Fractions containingP450_(OX), identified by their absorption at 420 nm, their CO bindingspectra and their ability to metabolize oxime in reconstitutionexperiments (see Example 4), are combined (approximately 200 ml). Theyare used for further purification or can be frozen. The combinedP450_(OX) fractions are adjusted during constant stirring to 30% (v/v)glycerol and 6% Triton X-114 by the dropwise addition of appropriateamounts of a mixture of glycerol and Triton X-114. The stirring iscontinued for 20 min, and is followed by 25 minutes of centrifugation at24500×g, 25° C., and no brake (temperature induced Triton X-114 phasepartitioning). Two phases are formed, a yellow upper phase and a clearlower phase. The lower phase which contains the major part of thecytochrome P450_(OX) activity is collected is diluted 2.5 fold toapproximately 350 ml with buffer C and applied with a flow rate of 70ml/h to a 1.9×5 cm column of Cibacron blue 3GA-agarose equilibrated inbuffer C. The column is washed with 50 ml of buffer C and the retainedcytochrome P450_(OX) is eluted with approximately 60 ml of a 0-1.5 M KCllinear gradient in buffer C. Teh fractions which by SDS-PAGE show thepresence of a single polypeptide band in the 50-60 kDa region arecombined and dialyzed under nitrogen for 24 h against 1 l of 8.9%glycerol, 10 mM KH₂PO₄/K₂HPO₄ (pH 7.9), 5 mM EDTA, 2 mM DTT (dialysisbuffer) to reduce the salt and detergent content. The enzyme preparationis frozen in liquid nitrogen, and stored at −80° C.

Example 4

[0080] Characterization of Cytochrome P450_(OX) Obtained by Isolationfrom Sorghum Microsomes

[0081] 4.1. Molecular Weight and Amino Acid Sequence Data

[0082] The molecular weight of P450_(OX) as determined by SDS-PAGE is 55kD. The protein band corresponding to the P450_(OX) isolated from theCibacron blue 3GA-agarose column is excised from 8-25%SDS-polyacrylamide gels and electroeluted. The electroeluted protein isdigested with endoproteinase Glu-C (protease V8 sequencing grade, 18 h,23° C.) according to the manufacturer (Boehringer Mannheim) using anapproximate 1:100 weight ratio between proteinase and protein. Theelectroeluted protein and the digested protein sample are subjected toSDS-PAGE, and the protein and fragments transferred to ProBlottmembranes (Applied Biosystems). Coomassie stained regions of themembrane are excised and subjected to N-terminal amino acid sequencingon an Applied Biosystems model 470A Sequenator equipped with an on-linemodel 120A phenylthiohydantoin amino acid analyzer.

[0083] N-terminal amino acid sequencing produced two sequences, whichcould be read independently due to their difference in relativeabundance. A database search (BLAST) showed the sequence -GLVKEGVDMEEGTLto differ in only a single position from the N-terminal sequence of theB subunit of the vacuolar ATPase of barley (Hordeum vulgare) which isMGLVKEGADMEEGTL (accession number L11862). The barley B subunit has apredicted molecular mass of 54 kDa (20). The presence of the B subunitof the vacuolar ATPase as a contaminant in the P450_(OX) preparation wasfurther substantiated by Western blotting which showed a single band at55 kDa when using a monoclonal antibody raised against the B subunit ofthe vacuolar ATPase from oat roots provided by Dr. Heven Sze. The Bsubunit could be depleted from the P450_(OX) preparation byimmobilization on antibody coated microtiter wells. This approachpermitted unambiguous determination of the N-terminal amino acidsequence of P450_(OX) as -ATTATPQLLGGSVPEQ and in addition provided thesequence of one internal P450_(OX) peptide fragment, MDRLVADLDRAAA.Attempts to remove the residual amounts of the B subunit of the vacuolarATPase resulted in the formation of carbon monoxide difference spectrain which the 420 nm component representing inactive the denatured P420form of P450_(OX) was largely increased and in loss or significantlydiminished ability to reconstitute the P450_(OX) activity in thefractions obtained. This reflects the immanent lability of P450_(OX).The B subunit of the vacuolar ATPase is not expected to possess any ofthe catalytic properties associated with P450_(OX). Accordingly, thepresence of the B subunit as a contaminant was accepted in the metabolicstudies of P450_(OX) reported below.

[0084] N-terminal Sequence:

[0085] -- A T T A T P Q L L G G S V P E Q -- (SEQ ID NO: 3)

[0086] Internal Sequence:

[0087] -- M D R L V A D L D R A A A -- (SEQ ID NO: 4)

[0088] 4.2. Isolation of the NADPH-P450 Oxidoreductase

[0089] The NADPH-P450 oxidoreductase binds to the DEAE-SepharoseFF/S-100-Sepharose column and is eluted by augmenting buffer A with 0.5M KCl. The reductase is subsequently purified to homogeneity on a columnof 2′,5′-ADP-Sepharose 4B (Pharmacia) as previously described (Halkierand Moller, Plant Physiol. 96:10-17, 1990) and concentrated toapproximately 15 units/ml.

[0090] 4.3. Preparation of Soluble UDPG Glucosyitransferase

[0091] The glucosyltransferase is partially purified by ammonium sulfatefractionation of the centrifugation supernatant obtained during thepreparation of microsomes. The glucosyltransferase fraction precipitatesbetween 40% and 60% (NH₄)₂SO₄ and is dissolved in 5 ml of 50 mM Tricine(pH 7.9), 2 mM DTT, and dialyzed against 2 l of the same bufferovernight.

[0092] 4.4. Reconstitution of Cytochrome P450_(OX) Activity

[0093] Reconstitution of the enzyme activity of a microsomal cytochromeP450 is accomplished by inserting the cytochrome P450 enzyme and thecorresponding NADPH cytochrome P450 oxidoreductase into lipid micelles.A mixture of lipids can be used but in the case of cytochrome P450_(OX),dilauroylphosphatidylcholine (DLPC) provides the best enzymaticactivity. The number of correctly formed complexes of cytochromeP450_(OX) and NADPH cytochrome P450 oxidoreductase are a rate limitingfactor. Excess amounts of the oxidoreductase and concentrated enzymesolutions are utilized to ensure a sufficient number of activecomplexes.

[0094] A functionally reconstituted enzyme is obtained using thefollowing components: Cytochrome P450_(ox): 20 μg/ml in dialysis bufferNADPH cytochrome P450 oxidoreduc- 100 μg/ml in 50 mM potassium tasepurified from Sorghum bicolor: phosphate buffer (pH 7.9) Lipid: 10 mg/mldilauroylphospha- tidylcholine, sonicated in 50 mM Tricine (pH 7.9)NADPH: 25 mg/ml in H₂O ¹⁴C-oxime, enzymatically produced 0.01 μCi/μl,394 mCi/mmol from [U-¹⁴C]-L-tyrosine using reconstituted P450_(TYR), andpurified on HPLC

[0095] 5 μl lipid suspension is mixed in an eppendorf tube with 5 μlNADPH cytochrome P450 oxidoreductase (0.075 units), 10 μl of thecytochrome P450_(OX) (approximately 0.4 pmol) solution, and 0.5 μl¹⁴C-oxime (0.014 μCi/μl, 394 mCi/mmol). The final volume is adjusted to30 μl using 50 mM Tricine (pH 7.9) and the enzyme reaction is initiatedby addition of 1 μl of NADPH solution. Control samples are prepared byeither omitting the NADPH cytochrome P450 oxidoreductase or NADPH fromthe reaction mixture. The tubes are incubated under constant and gentleagitation at 30° C. for 1 h. After incubation the reaction mixtures areapplied to silica coated TLC sheets (Silica gel 60 F₂₅₄, Merck) anddeveloped using an ethyl acetate/toluene (1:5 v/v) mixture as mobilephase. The sheets are placed on storage phosphor screens over night andthe resultant products, p-hydroxy phenylacetonitrile andp-hydroxybenzaldehyde are visualized using a STORM 840 phosphorimagerfrom Molecular Dynamics.

[0096] When reconstituted into lipid micelles cytochrome P450_(OX)catalyzes the conversion of p-hydroxyphenylacetaldehyde oxime top-hydroxymandelonitrile which dissociates to p-hydroxybenzaldehyde andHCN. This demonstrates that cytochrome P450_(OX) is a multifunctionalprotein catalyzing both the conversion of p-hydroxyphenylacetaldehydeoxime to p-hydroxyphenylacetonitrile, and the conversion ofp-hydroxyphenylacetonitrile to p-hydroxymandelonitrile. P450_(OX)activity is strictly dependent on the presence of NADPH-P450oxidoreductase and NADPH. Sodium dithionite (10 mM) does not supportmetabolism of p-hydroxyphenylacetaldoxime. Omission of dialysis of theenzyme prior to reconstitution causes a relative increase in theaccumulation of p-hydroxyphenylacetonitrile compared top-hydroxybenzaldehyde.

[0097] 4.5. In vitro Reconstitution of the Complete Pathway of DhurrinSynthesis from its Parent Amino Acid Tyrosine

[0098] The complete reaction mixtures contain: 3 μl of isolated,recombinant P450_(TYR) (6 pmol, heterologously expressed in E.coli andisolated as in Halkier et al, Arch. Biochem. Biophys. 322: 369-377,1995), 10 μl of isolated and dialyzed P450_(OX) (approximately 0.4pmol), 5 μl of NADPH-P450 oxidoreductase (0.075 U), 1 μl of partiallypurified UDPG glucosyl transferase from Sorghum, 5 μl of DLPC (10 mg/mlin 50 mM Kp_(i) (pH7)), 0.25 μl of [U-¹⁴C]-tyrosine (0.05 μCi/mmol, 443mCi/mmol, Amersham), 3 μl of UDPG (33 mg/ml in 50 mM Kp_(i) (pH7)), and3 μl of castanospermin (2 mM in 50 mM Kp_(i) (pH7)). The components aremixed by repeated suspension and if necessary the final volume adjustedto 30 μl by the use of 50 mM Kp_(i) (pH7). The enzyme reaction isinitiated with 1 μl of NADPH (25 mg/ml). Dhurrin is also synthesized viareconstitution of P450_(OX) with p-hydroxyphenylacetaldehyde oxime(leaving out P450_(TYR) and tyrosine from the reaction mixtures anyadditional components being unchanged.). These assays contain either 0.5μl of [U-¹⁴C]-p-hydroxyphenylacetaldehyd oxime (0.014 μCi/μl, 394mCi/mmol) or 3 μl of unlabelled p-hydroxyphenylacetaldehyde oxime (20mM) as substrate for P450_(OX). In the latter case the radioactive labelis 1 μl of [U-¹⁴C]-UDPG (0.025 μCi/μl, 287 mCi/mmol, Amersham). Allreaction mixtures are prepared as duplicates. After incubation for 1 hat 30° C. each set of reaction mixtures is applied to TLC sheets. Thefirst set of reaction mixtures is analyzed using the ethylacetate/toluene solvent as in example 4.5. The second set of reactionmixtures is analyzed using a solvent system consisting of ethylacetate/acetone/dichloromethane/methanol/water (20/15/6/5/4, v/v/v/v/v)in order to achieve separation of the hydrophilic product dhurrin fromtyrosine and from the hydrophobic intermediates. Radiolabelledsubstrates and products are visualized using the STORM840-phosphorimager.

[0099] The combined use of isolated P450_(TYR) and P450_(OX) inreconstitution experiments with radiolabeled tyrosine as substrateresults in the production of p-hydroxyphenylacetonitrile andp-hydroxybenzaldehyde. This demonstrates that P450_(TYR) and P450_(OX)are able to act together in vitro. The p-hydroxyphenylacetaldoximeproduced by P450_(TYR) is thus effectively used as a substrate byP450_(OX). No activity was observed in the absence of NADPH-P450oxidoreductase or in the absence of NADPH. In vitro production ofdhurrin using p-hydroxyphenylacetaldoxime as substrate was accomplishedby reconstitution of P450_(OX) together with partially purified solubleUDPG glucosyltransferase in the presence of NADPH and UDPG. A cDNA clonefrom sorghum encoding the UDPG glucosyltransferase which specificallyutilizes p-hydroxymandelonitrile as a substrate is not available.Accordingly, in the present study a crude extract of the soluble UDPGglucosyltransferase from sorghum was used to glucosylatep-hydroxymandelonitrile and to demonstrate the in vitro reconstitutionof the entire dhurrin biosynthetic pathway. The radiolabeledp-hydroxyphenylacetaldoxime applied was fully metabolized.Castanospermine was added to inhibit the glucosidase activity present inthe UDPG glucosyltransferase preparation. In addition to the TLC systemused above for separation of hydrophobic compounds, an additional TLCsystem was introduced for the separation of hydrophilic compounds likedhurrin. The p-hydroxymandelonitrile formed in the reconstitution assaywas partly converted to dhurrin as demonstrated by the formation of aradiolabeled compound comigrating with authentic dhurrin. The assignmentof this radiolabeled compound as dhurrin was further substantiated byits breakdown in the absence of castanospermine, and by the formation ofa comigrating radiolabeled product when the experiment was repeated withradiolabeled UDPG instead of radiolabeled p-hydroxyphenylacetaldoxime.The radiolabeled UDPG unspecifically labeled a range of relativelyhydrophilic compounds. Due to the lability of p-hydroxymandelonitrileits conversion to dhurrin is experimentally detected as a disappearanceof p-hydroxybenzaldehyde. When radiolabeled p-hydroxyphenyl-acetaldoximewas used as substrate, a number of unidentified, hydrophobic,radiolabeled compounds were produced in addition to dhurrin. Theformation of these compounds occurs in the absence of UDPG but requiresthe presence of the soluble extract, which indicates that the UDPGglucosyltransferase extract contains additional enzymatic activities.Glucosylation of the phenolic group of p-hydroxymandelonitrile wouldresult in the formation of p-glucopyranosyloxymandelonitrile. Noradiolabeled product comigrating with an authentic standard ofp-glucopyranosyloxymandelonitrile was observed. The glucosidase activitypresent in the UDPG glucosyltransferase extract was efficientlyinhibited by castanospermine.

[0100] Upon in vitro reconstitution, the turn-over number of P450_(TYR)(CYP79) is 230 min⁻¹ (Sibbesen et al, J. Biol. Chem. 270: 3506-3511,1995). The partial conversion of P450_(OX) into its denatured P420 formprevents determination of its turn-over number. Using the microsomalsystem, the K_(m) and V_(max) values for p-hydroxymandelonitrileproduction from tyrosine, p-hydroxyphenylacetaldoxime, andp-hydroxyphenylacetonitrile are 0.03, 0.05, and 0.10 mM, and 145, 400,and 50 nmoles mg protein⁻¹ h⁻¹, respectively (Møller et al, J. Biol.Chem. 254: 8575-8583, 1979).

[0101] The entire dhurrin biosynthetic pathway starting from its parentamino acid tyrosine was reconstituted in vitro by combining P450_(TYR),P450_(OX), NADPH-P450 oxidoreductase in DLPC micelles with UDPGglucosyltransferase, tyrosine, NADPH, UDPG, and castanospermine.Tyrosine is converted by P450_(TYR) to p-hydroxyphenylacetaldoxime,which is further converted to p-hydroxyphenylacetonitrile andp-hydroxybenzaldehyde by P450_(OX). Some p-hydroxyphenylacetonitrileaccumulates, whereas all the p-hydroxymandelonitrile formed is convertedto dhurrin and some unidentified compounds. In this set of experiments,the stoichiometric ratio between P450_(TYR) and P450_(OX) isapproximately 15. It is therefore not surprising to detect theaccumulation of the p-hydroxyphenylacetaldoxime in the reconstitutionassay. The observed accumulation of p-hydroxyphenylacetonitrile isunexpected since previous experiments with sorghum microsomes have shownthat p-hydroxyphenylacetonitrile is difficult to accumulate and trap.Partial denaturation or inactivation of the isolated P450_(OX) mayexplain why p-hydroxyphenylacetonitrile accumulates in thereconstitution experiments with isolated P450_(OX).

[0102] 4.6. Substrate Binding

[0103] The identification of P450_(OX) as a multifunctional enzymeconverting p-hydroxyphenylacetaldoxime to p-hydroxymandelonitrile withp-hydroxyphenylacetonitrile as an intermediate stimulated us toinvestigate the substrate binding ability of P450_(OX). A reverse type Ispectrum with an absorption minimum at 381 nm and an absorption maximumat 418 nm was obtained with p-hydroxyphenylacetaldoxime suggesting ashift from a high to a low spin state upon substrate addition. Theamplitude increased in size upon incubation and reached a stable maximumafter approximately 45 min. No substrate binding spectrum was obtainedupon the addition of p-hydroxyphenylacetonitrile.

[0104] P450_(OX) was found to be much more labile compared to other P450enzymes isolated from sorghum. The isolated P450_(OX) produces a reverseType I substrate binding spectrum upon incubation withp-hydroxyphenylacetaldoxime. The extinction coefficient E₄₂₀₋₃₉₀corresponding to a complete transition from one spin state to the otheris 130 mM⁻¹ cm⁻¹. In the substrate binding spectra obtained, the maximalamplitudes are approximately twice as large as theoretically calculatedeven when assuming a complete shift from a high spin to a low spinstate. This discrepancy indicates that the P450_(OX) concentration wasunderestimated when quantified from the 450 nm peak in the carbonmonoxide binding spectrum. Alternatively, the P420 form of P450_(OX) isable to bind the oxime and thus contributes to the size of the substratebinding spectrum formed. The latter possibility could explain whymaximal amplitudes are only obtained after prolonged incubation.

[0105] P450 mediated dehydration of aldoximes to nitriles has previouslybeen reported using liver microsomes (DeMaster et al, J. Org. Chem.5074-5075, 1992). A major difference between the liver microsomal systemand P450_(OX) is that the former requires strict anaerobic conditionswhereas the latter proceeds aerobically, catalyzes a subsequentC-hydroxylation reaction, and metabolizes the (e)- as well as the(Z)-isomer. Under anaerobic conditions, a weak Type I spectrum isobtained with the liver microsomes. Upon addition of NADPH ordithionite, a pronounced Soret peak at 442-444 nm is formed. This isconcluded to represent the key active species of the P450 in the Fe (II)state. Spectral investigations of P450_(OX) under anaerobic conditionsdid not disclose the formation of a 442 nm absorbing complex, but thepresence of NADPH is required for catalytic activity which indicatesthat P450_(OX) also needs to be in the Fe (II) state to mediate thedehydration reaction.

Example 5

[0106] A-type Cytochrome P450 Probe Generation

[0107] PCR was performed on plasmid DNA isolated from a unidirectionalplasmid cDNA library (Invitrogen) made from 1-2 cm high etiolatedseedlings of Sorghum bicolor (L) Moench using highly degenerated inosine(I) containing primers preferentially selecting for A-type cytochromesP450 (Nelson and Durst, Drug Metabolism and Drug Interactions 12:189-206 (1995)). Primer 1 (sense strand) with the sequence5′-GCGGAATTCTTYIIICCNGAR MGNTT-3′ (SEQ ID NO:5) covers the consensusamino acid sequence FXPERF (SEQ ID NO:6) where X is any amino acid.Primer 2 (antisense strand) with the sequence5′-GCGGATCCIIIRCAIIINCKNCKNCC-3′ (SEQ ID NO:7) covers the consensusamino acid sequence GRRXCXG (SEQ ID NO:8). Primer 1 and primer 2 weretailed with EcoRI and BamHI sites, respectively, to ensure that only PCRproducts generated from both primers were cloned in EcoRI/BamHI digestedpBluescript II SK (Strategene). PCR was performed in two consecutiverounds. Round 1 using primer 1 and standard T7 primer5′-AATACGACTCACTATAG-3′ (SEQ ID NO:9) enriches the pool of cDNA encodingA-type cytochromes P450. Round 2 including primer 1 and primer 2generated predominantly one band of approximately 100 bp specific forA-type cytochromes P450. The PCR reaction for round 1 was set up in atotal volume of 100 μl containing 5% DMSO, 200 μM dNTPs, 200 pmol ofprimer 1, 100 pmol of standard T7 primer, 2.5 units Taq DNA Polymerasein PCR buffer and 1 μl of 100 times diluted plasmid DNA from the cDNAlibrary. The PCR reaction for round 2 was set up in a total of 100 μlcontaining 5% DMSO, 200 μM dNTPs, 200 pmol of primer 1 and primer 2, 2.5units Taq DNA Polymerase in PCR buffer and 1 μl of product obtained fromPCR round 1. For both rounds of PCR, one cycle of 5 min at 95° C. wasfollowed by 35 cycles of 30 sec at 95° C., 1 min at 50° C., and 30 sec72° C. The approximate 100 bp product of PCR round 2 was excised from a2% agarose gel and reamplified prior to cloning into pBluescript. Of the19 clones sequenced, 10 had very high sequence identity on the aminoacid level to cinnamic acid hydroxylase (CYP 74) and were therefore notfurther studied. Sequence comparisons of the remaining 9 sequencesdivided these into two groups of 8 and 1 sequences and were denoted “12”and “7”, respectively. A sequence “12” gene specific primer locatedbetween primer 1 and primer 2: 5′-GCGGATCCGACTACTACGGCTCGC-3′ (SEQ IDNO:10) and primer 5′-GCGGATCCTTTTTTTTTTTTTTTTV-3′ (SEQ ID NO:11) bothtailed with BamHI were used to amplify a “12” gene specific fragment ofapproximately 500 bp from PCR round 1 and cloned into pBluescript.Similarly a gene specific fragment for “7” was obtained using the “7”gene specific primer 5′-GCGGATCCGACATCAAGGGCAGCG-3′ (SEQ ID NO:12) andprimer 5′-GCGGATCCTTTTTTTTTTTTTTTTV-3′(SEQ ID NO:11). Inserts werelabelled with Digoxigenin-11-dUTP (Boehringer Mannheim) by PCRamplification with standard T7 and T3 primers according to themanufacturers instructions and used to screen the cDNA library.

Example 6

[0108] Library Screening and DNA Sequencing

[0109] All filter hybridizations were done using the DIG system(Boehringer Mannheim). Colony lifts were prepared using nylon membranes(Boehringer Mannheim) and hybridized over night at 68° C. in 5×SSC, 0.1%N-lauroylsarcosine, 0.02% SDS, 1% Blocking Reagent (BoehringerMannheim). Filters were washed twice for 15 minutes in 0.1×SSC, 0.1%SDS, at 65° C. prior to detection. Full-length clones were obtained forboth “12” and “7” as evidenced by sequence analysis. Sequencing was doneusing the Thermo Sequenase Fluorescent labelled Primer cycle sequencingkit (7-deaza dGTP) (Amersham) and analyzed on an ALF-Express(Pharmacia). Sequence computer analysis was done using the programs inthe GCG Wisconsin SequenceAnalysis Package. The full-length cDNAsequence of P450_(OX) and the derived amino acid sequence of the codingregion as obtained from nucleotide sequencing of “12” are given in isgiven in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

Example 7

[0110] Expression in E. Coli

[0111] The expression vector pSP19g10L (Barnes, Methods in Enzymology272: 3-14, 1996) was obtained from Dr. Henry Barnes (SyntheticGenetics/Immune Complex Incorporation, San Diego, Calif.). This plasmidcontains the lacZ promoter fused to the short leader sequence of thegene 10 from T₇ bacteriophage, g10L, which has been documented as anexcellent leader sequence for the expression of various heterologousproteins (Olin et aL, 1988). “7” and “12” were modified by PCR usingPwo-polymerase (Boehringer Mannheim) to introduce a Ndel site at thestart codon and to change the stop codon to an ochre stop codonimmediately followed by a HindIII site. For generation of an expressionclone for “7” primer 3 (sense strand)5′-CGCGGATCCATATGGACGCATCATTACTCCTCTCCGTCGCGCTC-3′(SEQ ID NO:13) andprimer 4 (antisense strand) 5′-CGCAAGCTTATTACATCTCAAC GGGGACCCT-3′(SEQID NO:14) were used. Primer 3 introduces silent mutations in codons 3,4, and 5 to reduce the G/C content around the translation start site anda BamHI site immediately upstream of the NdeI site. The obtained PCRfragment was digested with BamHI and HindIII and ligated into BamHI andHindIII digested pBluescript and controlled by sequencing to exclude PCRerrors. Similarly “12” was introduced into pBluescript using primer 5(sense strand) 5′-CGCGGATCCATATGGCAACAACAGCAACCCCGCAGCTCCTC-3′(SEQ IDNO:15) and primer 6 (antisense strand) 5′-CGCAAGCTTATTATGCTGCGCGGCGGTTCTTGTATTTGG-3′ (SEQ ID NO:16). Primer 5 introduces silent mutationsin codons 2, 3, 4, and 5 and primer 6 introduces silent mutations in thelast 2 codons to reduce the G/C content. The inserts were cut out usingNdeI and HindIII and ligated into NdeI and HindIII digested pSP19g10L.Expression plasmids were transformed into E. coli JM109 cells. Singlecolonies were grown overnight in LB medium containing 100 mgampicillin/ml at 37° C., and 5 ml of the overnight culture used toinoculate 500 ml of TB medium containing 50 mg/ml ampicillin, 1 mMthiamine, 1 mM isopropyl -β-thiogalactopyranoside, and 1 mMδ-aminolevulinic acid. Cells were grown at 28° C. for 48 hours at 125rpm. 1 ml of E. coli transformed with expression constructs of “7”,“12”, and pSP19g10L were pelleted through centrifugation (2000 g, 10min), washed and concentrated 10 fold in 50 mM Tricine pH 7.9 andincubated with 14 nCi [U-¹⁴C] p-hydroxyphenylacetaldehyde oxime with aspecific activity of 394 mCi/mmol at 30° C. for 30 min. The incubationmixtures were extracted with ethyl acetat, applied to a TLC plate(Silica gel 60 F₂₅₄, Merck), developed using an ethyl acetate/toluene(1:5 v/v) mixture as mobile phase, and visualized using a STORM 840 fromMolecular Dynamics. E. coli transformed with the construct expressing“12” was able to convert p-hydroxyphenylacetaldehyde oxime intop-hydroxyphenylacetonitrile.

[0112] CO difference spectra of solubilized spheroblasts of E. coliexpressing P450_(OX) contained a major peak at 417 nm and a minor peakat 457 nm. Generally, a CO spectrum with an absorbance peak around 420nm is indicative of a cytochrome P450 in a non-functional conformation(Imai et al, Eur. J. Biochem. 1: 419-426, 1964). The presence of a majorpeak at 417 nm suggests that the majority of the expressed cytochromeP450 was present in a non-functional conformation. The apparent shift inabsorbance peak from 450 nm to 457 nm may be due to the presence oflarge amounts of cytochrome P450 in the non-conformational state. Basedon the peak at 457 nm, the production was estimated to be 50 nmol of ofP450_(OX) per liter E. coli culture per 65 hours.

[0113] 5 μl membranes isolated as described in Halkier et al, Archivesof Biochemistry and Biophysics 322: 369-377, 1995, from E. coliexpressing “12” was reconstituted with 0.225 units NADPH-cytochromeP450-reductase, 50 μg NADPH, 42 nCi p-hydroxyphenylacetaldehyde oxime,and 100 μg dilaurylphosphatidylcholine in a total volume of 100 μl of 30mM Tricine pH 7.9. After incubation at 30° C. for 30 minutes thereaction mixture was applied to a TLC plate and analyzed as describedabove. Reconstitution of membranes from E. coli expressing “12” resultedin the accumulation of p-hydroxybenzaldehyde which is the stabledissociation product of p-hydroxymandelonitrile, the last intermediatein the biosynthesis of the cyanogenic glucoside dhurrin. This shows thatdemonstrates that “12” is the cytochrome P450 that catalyzes theconversion of p-hydroxyphenylacetaldehyde oxime top-hydroxymandelonitrile. The cDNA is designated P450_(OX). A clonecomprising the described cDNA of P450_(OX) has been deposited on Jan.10, 1997 with the DSMZ-Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, under theaccession number DSM 11367.

[0114] When radioactively labeled phydroxyphenylacetaldoxime wasadministrated to E. coli cells transformed with P450_(OX),p-hydroxyphenylacetonitrile accumulated in the P450_(OX)-expressing E.coli cells. E. coli, which does not contain endogenous cytochromes P450or a NADPH-cytochrome P450-reductase, has been shown to support thecatalytic activity of heterologously expressed cytochromes P450. Twosoluble E. coli flavoproteins, flavodoxin and NADPH-flavodoxinreductase, donate the reducing equivalents to the recombinantcytochromes P450. Reconstitution of isolated E. coli membranes orpurified recombinant enzyme with sorghum NADPH-cytochrome P450-reductaseresults in the conversion of p-hydroxyphenylacetaldoxime top-hydroxymandelonitrile, whereas P450_(OX)-expressing E. coli cellsmetabolize p-hydroxyphenylacetaldoxime to onlyp-hydroxyphenylacetonitrile. The inability of E. coli to support theconversion of p-hydroxyphenylacetonitrile to p-hydroxymandelonitrile isthe first example that the E. coli flavodoxin/NADPH-flavodoxin reductasesystem is not able to support the catalytic activity of a microsomalcytochrome P450 reaction. Preliminary reconstitution experiments withthe membranous and soluble fractions of P450_(OX)-expressing E. colicells have shown that the soluble flavodoxin/NADPH-flavodoxin reductasesystem only supports P450_(OX) in the conversion ofp-hydroxyphenylacetaldoxime to p-hydroxyphenylacetonitrile, and that aninhibitory factor hampering the subsequent hydroxylation reaction is notpresent in the soluble fraction. The inability of E. coli to supportfull P450_(OX) activity might reflect the atypical catalytic reactivityof P450_(OX). In E. coli cells transformed with P450_(OX) or with theexpression vector a compound with a slightly lower mobility thanp-hydroxyphenylacetaldoxime accumulated. This shows that E. coli is ableto metabolize p-hydroxyphenylacetaldoxime independent of the presence ofP450_(OX).

Example 8

[0115] Purification of Recombinant P450_(OX) (CYP71E1)

[0116] Spheroblasts from 2 l E. coli expressing P450_(OX) were weresubjected to temperature-induced phase partitioning with 1% Triton X-114as previously described (Halkier et al, Archives of Biochemistry andBiophysics 322: 369-377, 1995). The P450_(OX) containing upper phase wasdiluted 100 fold in 10 mM KP_(i) pH 7.0, 0.05% reduced Triton X-100, 1mM DTT, 0.5 mM PMSF, the pH adjusted to 7.0 with acetic acid, andapplied to a 16 ml fast flow CM-Sepharose column (Pharmacia)equilibrated in buffer D (10 mM KP_(i) pH 7.0, 0.2% Triton X-114, 0.05%reduced Triton X-100, 10% glycerol, 1 mM DTT, 0.5% PMSF). The column waswashed in buffer D and P450_(OX) eluted with a 0-1 M KCl linear gradient(350 ml) in buffer D. The combined P450_(OX) containing fractions (43ml) were used for the reconstitution experiments and electroelutedrecombinant P450_(OX) was used for antibody production in chicken.Purified recombinant P450_(OX) reconstituted with NADPH-cytochromeP450-reductase in DLPC catalyses the conversion ofp-hydroxyphenylacetaldehyde oxime to p-hydroxy-mandelonitrile in thepresence of NADPH as described in example 4 section 4.2 for thepurification of the plant enzyme.

Example 9

[0117] Expression of Dhurrin in Transgenic Arabidopsis and Tobacco

[0118] 9.1 Construction of Vector Plasmids

[0119] Three binary vectors for Agrobacterium tumefaciens mediatedtransformation, namely pPZP111.79, pPZP221.71E1, and pPZP111.79.71E1,are generated. For the construction of pPZP111.79 a cDNA clone ofP450_(TYR) (WO 95/16041; Koch et al, Arch Biochem Biophys 323:177-186,1995) is first excised with EcoRI and introduced into the EcoRI site ofpRT101 (Kopfer et al, Nucleic Acids Research 15: 5890, 1987) tofunctionally join the cDNA to the 35S-promoter and a CaMVpolyadenylation signal generating plasmid pRT101.79. Prior to theintroduction of the P450_(TYR) cDNA a part of the pRT101 polylinker isremoved by digestion with SacI and XbaI followed by religation of theblunt-ended ends obtained by Klenow treatment, thus leaving only theEcoRI and XhoI sites available. P450_(TYR) including the 35S-promoterand CaMV polyadenylation signal is excised from pRT101.79 using SphI. Togenerate plasmid pPZP111.79 the ends are blunt-ended with Klenowpolymerase and the fragment obtained is ligated into EcoRI cut plasmidpPZP111 (Hajdukiewicz et al, Plant Mol Biol 25: 989-994, 1994), the endsof which have been blunt-ended with Klenow polymerase, too, anddephosphorylated.

[0120] For the construction of pPZP221.71E1 the cDNA clone of P450_(OX)is first excised with KpnI and XbaI and ligated into the KpnI and XbaIsites of pRT101, generating pRT101.71E1. Subsequently P450_(OX)including the 35S-promoter and CaMV polyadenylation signal is excisedfrom pRT101.71E1 with HindIII and ligated into the HindIII site ofpPZP221 (Hajdukiewicz et al, Plant Mol Biol 25: 989-994, 1994) thusgenerating pPZP221.71E1. For the construction of pPZP111.79.71E1 thecDNA clone of P450_(OX) including the 35S-promoter and the CaMVpolyadenylation signal is excised from pPZP221.71E1 using HindIII,blunt-ended with Klenow polymerase and ligated into the SmaI site ofpPZP111.79.

[0121] 9.2 Transformation of Arabidopsis thaliana

[0122] The binary vectors pPZP111, pPZP221, pPZP111.79, pPZP221.71E1,and pPZP111.79.71E1 are introduced into Agrobacterium tumefaciens strainC58C1/pGV3850 by electroporation as described by Wenjun and Forde,Nucleic Acids Research 17: 8385, 1989. Arabidopsis thaliana ecotypeColombia is transformed by vacuum infiltration essentially as describedby the method of Bechtold et al, Molecular Biology and Genetics 316:1194-1199, 1993. Transformants are selected on MS plates containingeither 50 μg/ml kanamycin for the pPZP111 vector series, or 200 μg/mlgentamycin sulfate for the pPZP221 vector series. 4 to 6 weeks aftergermination kanamycin or gentamycin resistant plants are transferred tosoil.

[0123] 9.3 Transformation of Nicotiana tabacum cv Xhanti

[0124]Nicotiana tabacum cv Xhanti is transformed essentially by the leafdisc method of Svab et al (Methods in Plant Molecular Biology, ColdSpring Harbor, pp. 55-60, 1995) using Agrobacterium tumefaciensC58C1/pGV3850 transformed with either pPZP111.79, pPZP221.71E1, orpPZP111.79.71E1. 100 μg/ml kanamycin is used for selection oftransformants with the pPZP111.79 vector, 100 μg/ml gentamycin sulfatefor selection of transformants with the pPZP221.71E1 vector, and 50μg/ml G-418 for selection of transformants with the pPZP111.79.71E1vector. After rooting the tobacco plants are transferred to soil andgrown in a greenhouse.

[0125] 9.4 Determination of Dhurrin

[0126] The dhurrin content is quantified using the spectrophotometriccyanide assay previously described by Halkier et al (Plant Physiol 90:1552-1559, 1989) except that 5-10 mg of leaf tissue is frozen and thawedthree times before adding 0.1 mg β-d-glucosidase Type II (Sigma).

[0127] 9.5 Analysis of Transgenic Arabidopsis thaliana

[0128] Detached leaves of A. thaliana are fed 2 μl of (U-14C)-tyrosine(0.05 μCi/μl, 443 mCi/mmol, Amersham), and left over night in 100 μl H₂Oin closed eppendorf tubes. Metabolites are extracted with boiling 90%methanol for 5 min, the extracts concentrated, and applied to TLC sheets(Silica gel 60 F₂₅₄,). Metabolites are separated in three differentsolvent systems depending on their hydrophobicity/hydrophilicity. Thesolvent system ethyl acetate/acetone/dichloromethane/methanol/water(20/15/6/5/4, v/v/v/v/v) preferentially separates the differentcyanogenic glucosides. The solvent system isopropanol/ethylacetate/water (7/1/2) separates the different glucosinolates. Thesolvent system toluene/ethyl acetate (5/1) separates the hydrophobicintermediates. Radiolabelled substrates and products are visualizedusing a STORM 840 phosphorimager (Molecular Dynamics, USA).

[0129] Methanol extracts of A. thaliana leaves analyzed by TLC of plantstransformed with both P450_(TYR) and P450_(OX) using A. tumefaciensC58C1/pGV3850/pPZP111.79.71E1 reveals the presence of a new compoundthat co-migrates with the cyanogenic glucoside dhurrin. Feedingradiolabelled tyrosine to detached leaves shows that the radiolabel iscontained in the band that co-migrates with dhurrin. Analysis of wholeleaf tissue of the transformed plants by a colorimetric cyanide assay(Lambert et al., 1975, Analytic Chemistry 47, 917-919) reveals that theT1 transformants contained between 1 and 6 nmol cyanide/mg fresh weight.In comparison, control plants only transformed with nptII using A.tumefaciens C58C1/pGV3850/pPZP111 showed a cyanide content of 1.2±0.35nmol cyanide/mg fresh weight. Wild-type plants of A. thaliana have notbeen reported to contain cyanogenic glucosides, and the apparent levelsof cyanide detected in the control plants most likely reflects thepresence of thiocyanates. Thiocyanates are breakdown products ofglucosinolates, and are known to give a false reaction in thecolorimetric cyanide assay (Epstein, 1974, Analytic Chemistry 19,272-274). The observed production of large amounts of the cyanogenicglucoside dhurrin as a result of the introduction of both P450_(TYR) andP450_(OX) as exemplified with A. thaliana demonstrates that a suitableUDP-glucosyltransferase is present which glucosylates thep-hydroxymandelonitrile formed in the correct position. Since thestereospecificity of the glycosyltransferase is not known thepossibility exists that the cyanogenic glucoside produced is actuallytaxiphyllin which is the epimer (mirror image isomer) of dhurrin.

[0130] When P450_(TYR) is introduced by A. tumefaciensC58C1/pGV3850/pPZP111.79 into A. thaliana large quantities of a compoundthat co-migrates with the tyrosine derived glucosinolate p-hydroxybenzylglucosinolate accumulates. This documents that the introduction ofP450_(TYR) results in the generation of p-hydroxyphenylacetaldoxime fromtyrosine. The tyrosine derived oxime is then further metabolized by theenzymes in the glucosinolate pathway to p-hydroxybenzyl glucosinolate.This strongly indicates that the enzymes downstream of the oxime in theglucosinolate pathway have a low substrate specificity with respect tothe structure of the side chain and that the glucosinolate profile ingeneral is determined by the substrate specificity of the firstcytochrome P450 in the pathway.

[0131] The substrate specificity of P450_(OX) is not as narrow as thatof P450_(TYR). P450_(OX) can metabolize other amino acid derived oximesas exemplified by the phenylalanine derived oxime, phenylacetaldoxime,whereas P450_(TYR) can only metabolize tyrosine. By introducingP450_(OX) into glucosinolate producing plants, it can therefore beexpected that cyanogenic glucosides accumulate as generated from aminoacid derived oximes in the glucosinolate biosynthetic pathway.

[0132] Because P450_(TYR) and P450_(OX) interact with the enzymes andthe precursors in the glucosinolate biosynthetic pathway it is expectedthat the glucosinolate profile will also be altered.

[0133] 9.6 Analysis of Transgenic Nicotiana tabacum cv Xhanti

[0134] Microsomes isolated from tobacco plants transformed withpPZP111.79 and functionally expressing CYP79 catalyze the formation oftyrosine to p-hydroxyphenylacetaldoxime. Methanol extracts from detachedtobacco leaves expressing CYP79 and fed radioactive labeled tyrosinecontain three additional labeled bands compared to wild-type tobaccoplants when analyzed by TLC using the solvent system isopropanol/ethylacetate/water (7/1/2). The additional three bands co-migrate on theTLC's with labeled bands detected in wild-type tobacco plants fedradioactive labeled p-zydroxyphenylacetaldoxime. When analyzed in thesolvent system toluene/ethyl acetate (5/1) one of the bands is confirmedto be p-hydroxyphenylacetaldoxime as evidenced by co-migration with coldstandards. The identity of the two additional bands is not yet know, butfrom their mobility in the two solvent systems they are judged to beless hydrophobic than p-hydroxyphenylacetaldoxime, and most likely theyare glycosylated derivatives of p-hydroxyphenylacetaldoxime. Analyzes ofthe CYP79 plants confirms that CYP79 can be expressed functionally intobacco and that some free p-hydroxyphenylacetaldoxime is generated, butthat the majority of the p-hydroxyphenylacetaldoxime generated isfurther metabolized by the plants. Plants expressing both P450_(TYR) andP450_(OX) can be obtained by crossing plant expressing P450_(TYR) withplants expressing P450_(OX). Alternatively, plants expressing P450_(TYR)or P450_(OX) can be re-transformed with A. tumefaciensC58C1/pGV3850/pPZP221.71E1 or A. tumefaciens C58C1/pGV3850/pPZP111.79taking advantage of the fact that the two cytochromes P450 constructsare linked to the two different non exclusive resistant markers, nptIIand aacCI.

Example 10

[0135] Expression of Dhurrin in Transgenic Maize

[0136] 10.1 Construction of Vector Plasmids

[0137] The following two vectors, pCIB 9842 and pCIB 9833, are generatedfor biolistic transformation of maize with P450_(TYR) and P450_(OX).

[0138] pCIB 9842: A cDNA clone encoding P450_(TYR) cloned into the EcoRIsite of pBluescript II SK as described in WO 95/16041 is used togenerate a BamHI site at the start ATG codon and a Bgl II site at thestop codon by PCR. The BamHI-Bgl II fragment containing the P450_(TYR)gene is cloned into BamHI and Bgl II cut pCIB 9805, a pUC19 based plantexpression vector engineered with AfIII/NotI/AscI sites 256 base pairsupstream from the the HindIII site and 778 bp downstream from the BgIIIsite and containing the metallothionin-like promoter disclosed inEP-A-452269 and the 35S terminator.

[0139] pCIB 9833: The P450_(OX) cDNA clone of Example cloned into aNotI-BstXI site of pcDNAII (Invitrogen) is used to generate a BamHI siteat the start ATG codon and a Bgl II site at the stop codon. TheBamHI-Bgl II fragment containing the P450_(OX) gene is cloned into pCIB9805 cut with BamHI and Bgl II, too.

[0140] 10.2 Methods of Transformation of Maize

[0141] Type I embryogenic callus cultures (Green et al, 1983; Wan et al,1994) of a Lancaster-type inbred are initiated from immature embryos,1.5-2.5 mm in length. Embryos are aseptically excised fromsurface-sterilized, greenhous-grown ears approximately 14 days afterpollination, placed on Duncan's callus initiation medium with 2% sucroseand 5 mg/l chloramben, and cultured in the dark. Embryogenic responsesare removed from the explants after about 14 days and placed ontoDuncan's maintenance medium with 2% sucrose and 0.5 mg/l 2,4-d. After 4to 8 weeks of weekly subculture to fresh maintenance medium, highquality compact embryogenic cultures are established. Actively growingembryogenic callus pieces are selected as target tissue for genedelivery. The callus pieces are plated onto target plates containingmaintenance medium with 12% sucrose approximately 4 hours prior to genedelivery. The callus pieces are arranged in circles, 8 and 10 mm fromthe center of the target plate. Plasmid DNA is precipitated onto goldmicrocarriers as described in the DuPont Biolistics manual. Two to threeμg of each plasmid is used in each 6 shot microcarrier preparation.Genes are delivered to the target tissue cells using the PDS-1000HeBiolistics device. The settings on the Biolistics device were asfollows: 8 mm between the rupture disc and the macrocarrier, 10 mmbetween the macrocarrier and the stopping screen and 7 cm between thestopping screen and the target. Each target plate is shot twice using650 psi rupture discs. A 200×200 stainless steel mesh (McMaster-Carr,New Brunswick, N.J.) is placed between the stopping screen and thetarget tissue. Seven days after gene delivery, target tissue pieces aretransferred from the high osmotic medium to selection medium containing100-120 mg/l glufosinate ammonium (Basta). All amino acids are removedfrom the selection medium. After 5 to 8 weeks on high level selectionmedium, any growing colonies are subcultured to medium containing 20mg/l Basta. The embryogenic callus is subcultured every 2 weeks for 4 to8 weeks and then transferred to a modified MS medium containing 3%sucrose, 0.25 mg/l ancymidol, 0.5 mg/l kinetin and no selection agentand placed in the light. Ancymidol and kinetin are removed after 2weeks. Regenerating shoots with or without roots are transferred toMagenta boxes containing MS medium with 3% sucrose and small plants withroots are eventually recovered and transferred to soil in thegreenhouse.

Example 11

[0142] Identification of P450_(TYR) Homologues in GlucosinolateContaining Species by PCR

[0143] Based on a computer sequence alignment of an ArabidopsisP450_(TYR) homologue EST (accession number T42902) and a P450_(TYR)homologue from Sinapis two degenerate primer oligonucleotides aredesigned which allow to amplify PCR fragments of P450_(TYR) homologuesfrom genomic DNA of glucosinolate containing species. Sense strandprimer (5′-GCGGAATTCAARCCIGARMGICAYYT-3′) covers the conserved aminoacid sequence KPERHL (SEQ ID NO: 18) and includes an EcoRI cloning site.Antisense strand primer 2 (5′-GCGGATCCRCAICCICKYTTICCNGT-3′) covers theconserved amino acid sequence TGKRGC (SEQ ID NO: 19) and includes aBamHI cloning site. PCR is performed on genomic DNA prepared with theNucleon Phytopure Plant DNA Extraction kit of Amersham. PCR reactionsare set up in a total volume of 100 μl containing 5% DMSO, 200 μM dNTPs,200 pmol of each primer, 2.5 units Taq polymerase in PCR buffer (50 mMKCl, 10 mM Tris-HCl (pH 8.8), 1.5 mM MgCl₂, and 0.1% Triton X-100) using1 μg of genomic DNA from either Sinapis alba, A. thaliana, Brassicanapus, Tropaeolum majus, or N. tabacum cv Xhanti. PCR is performed usingfour sequential stages:

[0144] stage 1: one cycle of 5 min at 95° C.;

[0145] stage 2: 5 cycles of 30 s at 95° C., 30 s at 55° C., 30 s at 72°C.;

[0146] stage 3: 30 cycles of 30 s at 95° C., 30 s at 60° C., 30 s at 72°C.; and

[0147] stage 4: one cycle of 5 min at 72° C.

[0148] To generate sufficient amounts of the approximately 100 bp bandfrom T. majus, stage 2 can be modified to 5 cycles of 30 s at 95° C., 30s at 50° C., 30 s at 71° C. PCR products are purified using the QIAquickPCR Purification Kit (Qiagen), restriction digested with EcoRI and BamHIand separated on a 3% TAE agarose gel. The dominant approximately 100 bpband is excised, and ligated into EcoRI/BamHI linearized pBluescript IISK (Stratagene). Approximately 10 clones from each of the 5 species aresequenced using the Thermo Sequence Fluorescent-labelled Primer cyclesequencing kit (7-deaza dGTP) (Amersham) and analyzed on an ALF-Express(Pharmacia).

[0149] From the four glucosinolate containing species S. alba, A.thaliana, B. napus, and T. majus PCR fragments encoding the conservedamino acid sequence, KPERH(L/F)NECSEVTLT ENDLRFISFSTGKRGC (SEQ ID NOs:20 and 21, respectively) are identified. This consensus amino acidsequence is identical to the P450_(TYR) homologue sequences from S. albaand A. thaliana previously identified and highly similar to the sorghumP450_(TYR) amino acid sequence. From the non-glucosinolate containingplant N. tabacum cv Xhanti a PCR fragment encoding this consensussequence could not be identified. The presence of this P450_(TYR)homologue consensus amino acid sequence in the exemplified fourglucosinolate containing plant species indicates that an amino acidN-hydroxylase cytochrome P450 of the P450_(TYR) family converts theparent amino acids or chain elongated parent amino acids into thecorresponding oximes in glucosinolate species. The generation of PCRfragments specific for the P450_(TYR) homologues allow the isolation ofhomologous cDNA or genomic clones from corresponding libraries.

[0150] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1 23 1929 base pairs nucleic acid double linear cDNA P450ox CDS 81..16731 CAGAGCTAGA AGCAGCTCAC ACTCCACACT CGTCTCGCCC GGCCATACCC CAAGGCAAGC 60AGGGGCACGG GCAATTAACA ATG GCC ACC ACC GCC ACC CCG CAG CTC CTC 110 MetAla Thr Thr Ala Thr Pro Gln Leu Leu 1 5 10 GGC GGC AGC GTG CCG CAG CAGTGG CAG ACG TGC CTC CTG GTG CTC CTC 158 Gly Gly Ser Val Pro Gln Gln TrpGln Thr Cys Leu Leu Val Leu Leu 15 20 25 CCT GTG CTG CTG GTG TCC TAC TACCTC CTC ACC AGC AGG AGC AGG AAC 206 Pro Val Leu Leu Val Ser Tyr Tyr LeuLeu Thr Ser Arg Ser Arg Asn 30 35 40 AGG AGC AGG AGC GGC AAG CTG GGC GGGGCG CCG CGG CTG CCG CCG GGC 254 Arg Ser Arg Ser Gly Lys Leu Gly Gly AlaPro Arg Leu Pro Pro Gly 45 50 55 CCT GCG CAG CTG CCG ATC CTG GGC AAC CTGCAC CTG CTG GGC CCG CTG 302 Pro Ala Gln Leu Pro Ile Leu Gly Asn Leu HisLeu Leu Gly Pro Leu 60 65 70 CCG CAC AAG AAC CTC CGC GAG CTG GCG CGG CGGTAC GGC CCC GTG ATG 350 Pro His Lys Asn Leu Arg Glu Leu Ala Arg Arg TyrGly Pro Val Met 75 80 85 90 CAG CTC CGT CTA GGC ACC GTG CCG ACG GTG GTGGTG TCC AGC GCG GAG 398 Gln Leu Arg Leu Gly Thr Val Pro Thr Val Val ValSer Ser Ala Glu 95 100 105 GCG GCG CGG GAG GTT CTC AAG GTG CAC GAC GTCGAC TGC TGC AGC CGG 446 Ala Ala Arg Glu Val Leu Lys Val His Asp Val AspCys Cys Ser Arg 110 115 120 CCG GCG TCG CCC GGT CCC AAG CGC CTC TCC TACGAC CTC AAG AAC GTC 494 Pro Ala Ser Pro Gly Pro Lys Arg Leu Ser Tyr AspLeu Lys Asn Val 125 130 135 GGC TTC GCG CCC TAC GGC GAG TAC TGG CGC GAGATG CGC AAG CTC TTC 542 Gly Phe Ala Pro Tyr Gly Glu Tyr Trp Arg Glu MetArg Lys Leu Phe 140 145 150 GCG CTC GAG CTC CTC AGC ATG CGC CGC GTC AAGGCC GCC TGC TAC GCG 590 Ala Leu Glu Leu Leu Ser Met Arg Arg Val Lys AlaAla Cys Tyr Ala 155 160 165 170 CGC GAG CAG GAG ATG GAC AGG CTC GTC GCCGAC CTC GAC CGC GCC GCC 638 Arg Glu Gln Glu Met Asp Arg Leu Val Ala AspLeu Asp Arg Ala Ala 175 180 185 GCG TCC AAG GCC TCC ATC GTC CTC AAC GACCAC GTC TTC GCC CTC ACC 686 Ala Ser Lys Ala Ser Ile Val Leu Asn Asp HisVal Phe Ala Leu Thr 190 195 200 GAC GGC ATC ATC GGC ACC GTC GCG TTC GGCAAC ATC TAC GCC TCC AAG 734 Asp Gly Ile Ile Gly Thr Val Ala Phe Gly AsnIle Tyr Ala Ser Lys 205 210 215 CAG TTC GCG CAC AAG GAG CGC TTC CAG CACGTG CTG GAC GAC GCC ATG 782 Gln Phe Ala His Lys Glu Arg Phe Gln His ValLeu Asp Asp Ala Met 220 225 230 GAC ATG ATG GCC AGC TTC TCC GCC GAG GACTTC TTC CCC AAC GCC GCC 830 Asp Met Met Ala Ser Phe Ser Ala Glu Asp PhePhe Pro Asn Ala Ala 235 240 245 250 GGC CGC CTC GCC GAC CGC CTC TCG GGCTTC CTC GCC CGC CGC GAG CGC 878 Gly Arg Leu Ala Asp Arg Leu Ser Gly PheLeu Ala Arg Arg Glu Arg 255 260 265 ATC TTC AAC GAG CTC GAC GTC TTC TTCGAG AAG GTC ATC GAC CAG CAC 926 Ile Phe Asn Glu Leu Asp Val Phe Phe GluLys Val Ile Asp Gln His 270 275 280 ATG GAC CCG GCG CGC CCC GTG CCG GACAAC GGC GGC GAC CTC GTC GAC 974 Met Asp Pro Ala Arg Pro Val Pro Asp AsnGly Gly Asp Leu Val Asp 285 290 295 GTC CTC ATC AAC CTG TGC AAG GAG CACGAC GGC ACG CTC CGC TTC ACC 1022 Val Leu Ile Asn Leu Cys Lys Glu His AspGly Thr Leu Arg Phe Thr 300 305 310 AGG GAC CAC GTC AAG GCC ATC GTC CTCGAC ACC TTC ATC GGC GCC ATC 1070 Arg Asp His Val Lys Ala Ile Val Leu AspThr Phe Ile Gly Ala Ile 315 320 325 330 GAC ACC AGC TCC GTC ACC ATC CTGTGG GCC ATG TCG GAG CTG ATG CGG 1118 Asp Thr Ser Ser Val Thr Ile Leu TrpAla Met Ser Glu Leu Met Arg 335 340 345 AAG CCG CAG GTG CTG AGG AAG GCGCAG GCC GAG GTG CGG GCC GCC GTG 1166 Lys Pro Gln Val Leu Arg Lys Ala GlnAla Glu Val Arg Ala Ala Val 350 355 360 GGC GAC GAC AAG CCG CGC GTC AACTCG GAA GAC GCC GCC AAG ATC CCG 1214 Gly Asp Asp Lys Pro Arg Val Asn SerGlu Asp Ala Ala Lys Ile Pro 365 370 375 TAC CTG AAG ATG GTG GTC AAG GAGACG CTG CGG CTG CAC CCG CCG GCG 1262 Tyr Leu Lys Met Val Val Lys Glu ThrLeu Arg Leu His Pro Pro Ala 380 385 390 ACG CTG CTG GTG CCC CGG GAG ACGATG CGG GAC ACC ACC ATC TGC GGC 1310 Thr Leu Leu Val Pro Arg Glu Thr MetArg Asp Thr Thr Ile Cys Gly 395 400 405 410 TAC GAC GTG CCG GCC AAC ACGCGC GTC TTC GTC AAC GCC TGG GCC ATC 1358 Tyr Asp Val Pro Ala Asn Thr ArgVal Phe Val Asn Ala Trp Ala Ile 415 420 425 GGC AGG GAC CCG GCG AGC TGGCCG GCG CCC GAC GAG TTC AAC CCG GAC 1406 Gly Arg Asp Pro Ala Ser Trp ProAla Pro Asp Glu Phe Asn Pro Asp 430 435 440 CGC TTC GTG GGG AGC GAC GTCGAC TAC TAC GGC TCG CAC TTC GAG CTC 1454 Arg Phe Val Gly Ser Asp Val AspTyr Tyr Gly Ser His Phe Glu Leu 445 450 455 ATA CCG TTC GGG GCC GGC CGCCGG ATC TGC CCG GGA CTC ACC ATG GGC 1502 Ile Pro Phe Gly Ala Gly Arg ArgIle Cys Pro Gly Leu Thr Met Gly 460 465 470 GAG ACC AAC GTC ACC TTC ACCCTC GCC AAC CTG CTC TAC TGC TAC GAC 1550 Glu Thr Asn Val Thr Phe Thr LeuAla Asn Leu Leu Tyr Cys Tyr Asp 475 480 485 490 TGG GCG CTG CCG GGG GCCATG AAG CCG GAG GAC GTC AGC ATG GAG GAG 1598 Trp Ala Leu Pro Gly Ala MetLys Pro Glu Asp Val Ser Met Glu Glu 495 500 505 ACC GGA GCG CTC ACG TTCCAC CGG AAG ACG CCG CTT GTG GTG GTG CCC 1646 Thr Gly Ala Leu Thr Phe HisArg Lys Thr Pro Leu Val Val Val Pro 510 515 520 ACC AAA TAC AAG AAC CGCCGC GCC GCC TAGTGAGCAG AGCCGAGCAG 1693 Thr Lys Tyr Lys Asn Arg Arg AlaAla 525 530 AGCAATGGTC GACGACGACG ACGACGACGA CTGAATAAGC GTGCCAAAGTTTAGTACT 1753 GTACGTACGT ACCTACTGCT ACTACGTACA GCTAGCCAAC AGTCAGAGTTGGACACTG 1813 GGAGCTATCA TCCGGTCCTC TTCTTTTTGT GATACGTATT TGTTATGTGTTTTAGTGC 1873 CAAAGCACAA AAGAAATAAA GCCCATCACA GTCGCGAGTC AAAAAAAAAAAAAAAA 1929 531 amino acids amino acid linear protein 2 Met Ala Thr ThrAla Thr Pro Gln Leu Leu Gly Gly Ser Val Pro Gln 1 5 10 15 Gln Trp GlnThr Cys Leu Leu Val Leu Leu Pro Val Leu Leu Val Ser 20 25 30 Tyr Tyr LeuLeu Thr Ser Arg Ser Arg Asn Arg Ser Arg Ser Gly Lys 35 40 45 Leu Gly GlyAla Pro Arg Leu Pro Pro Gly Pro Ala Gln Leu Pro Ile 50 55 60 Leu Gly AsnLeu His Leu Leu Gly Pro Leu Pro His Lys Asn Leu Arg 65 70 75 80 Glu LeuAla Arg Arg Tyr Gly Pro Val Met Gln Leu Arg Leu Gly Thr 85 90 95 Val ProThr Val Val Val Ser Ser Ala Glu Ala Ala Arg Glu Val Leu 100 105 110 LysVal His Asp Val Asp Cys Cys Ser Arg Pro Ala Ser Pro Gly Pro 115 120 125Lys Arg Leu Ser Tyr Asp Leu Lys Asn Val Gly Phe Ala Pro Tyr Gly 130 135140 Glu Tyr Trp Arg Glu Met Arg Lys Leu Phe Ala Leu Glu Leu Leu Ser 145150 155 160 Met Arg Arg Val Lys Ala Ala Cys Tyr Ala Arg Glu Gln Glu MetAsp 165 170 175 Arg Leu Val Ala Asp Leu Asp Arg Ala Ala Ala Ser Lys AlaSer Ile 180 185 190 Val Leu Asn Asp His Val Phe Ala Leu Thr Asp Gly IleIle Gly Thr 195 200 205 Val Ala Phe Gly Asn Ile Tyr Ala Ser Lys Gln PheAla His Lys Glu 210 215 220 Arg Phe Gln His Val Leu Asp Asp Ala Met AspMet Met Ala Ser Phe 225 230 235 240 Ser Ala Glu Asp Phe Phe Pro Asn AlaAla Gly Arg Leu Ala Asp Arg 245 250 255 Leu Ser Gly Phe Leu Ala Arg ArgGlu Arg Ile Phe Asn Glu Leu Asp 260 265 270 Val Phe Phe Glu Lys Val IleAsp Gln His Met Asp Pro Ala Arg Pro 275 280 285 Val Pro Asp Asn Gly GlyAsp Leu Val Asp Val Leu Ile Asn Leu Cys 290 295 300 Lys Glu His Asp GlyThr Leu Arg Phe Thr Arg Asp His Val Lys Ala 305 310 315 320 Ile Val LeuAsp Thr Phe Ile Gly Ala Ile Asp Thr Ser Ser Val Thr 325 330 335 Ile LeuTrp Ala Met Ser Glu Leu Met Arg Lys Pro Gln Val Leu Arg 340 345 350 LysAla Gln Ala Glu Val Arg Ala Ala Val Gly Asp Asp Lys Pro Arg 355 360 365Val Asn Ser Glu Asp Ala Ala Lys Ile Pro Tyr Leu Lys Met Val Val 370 375380 Lys Glu Thr Leu Arg Leu His Pro Pro Ala Thr Leu Leu Val Pro Arg 385390 395 400 Glu Thr Met Arg Asp Thr Thr Ile Cys Gly Tyr Asp Val Pro AlaAsn 405 410 415 Thr Arg Val Phe Val Asn Ala Trp Ala Ile Gly Arg Asp ProAla Ser 420 425 430 Trp Pro Ala Pro Asp Glu Phe Asn Pro Asp Arg Phe ValGly Ser Asp 435 440 445 Val Asp Tyr Tyr Gly Ser His Phe Glu Leu Ile ProPhe Gly Ala Gly 450 455 460 Arg Arg Ile Cys Pro Gly Leu Thr Met Gly GluThr Asn Val Thr Phe 465 470 475 480 Thr Leu Ala Asn Leu Leu Tyr Cys TyrAsp Trp Ala Leu Pro Gly Ala 485 490 495 Met Lys Pro Glu Asp Val Ser MetGlu Glu Thr Gly Ala Leu Thr Phe 500 505 510 His Arg Lys Thr Pro Leu ValVal Val Pro Thr Lys Tyr Lys Asn Arg 515 520 525 Arg Ala Ala 530 16 aminoacids amino acid unknown linear peptide N-terminal N-terminal peptide 3Ala Thr Thr Ala Thr Pro Gln Leu Leu Gly Gly Ser Val Pro Glu Gln 1 5 1015 13 amino acids amino acid unknown linear peptide internal Internalpeptide 4 Met Asp Arg Leu Val Ala Asp Leu Asp Arg Ala Ala Ala 1 5 10 26base pairs nucleic acid single linear DNA (genomic) Primer 1misc_feature 13..15 /note= “N = inosine” 5 GCGGAATTCT TYNNNCCNGA RMGNTT26 6 amino acids amino acid unknown linear peptide internal Amino acidsencoded by primer 1 6 Phe Xaa Pro Glu Arg Phe 1 5 26 base pairs nucleicacid single linear DNA (genomic) Primer 2 misc_feature 9..11 /note= “N =inosine” misc_feature 15..17 /note= “N = inosine” 7 GCGGATCCNNNRCANNNNCK NCKNCC 26 7 amino acids amino acid unknown linear peptideinternal Amino acids encoded by primer 2 8 Gly Arg Arg Xaa Cys Xaa Gly 15 17 base pairs nucleic acid single linear DNA (genomic) T7 primer 9AATACGACTC ACTATAG 17 24 base pairs nucleic acid single linear DNA(genomic) “12” gene specific primer 10 GCGGATCCGA CTACTACGGC TCGC 24 25base pairs nucleic acid single linear DNA (genomic) 11 GCGGATCCTTTTTTTTTTTT TTTTV 25 24 base pairs nucleic acid single linear DNA(genomic) “7” gene specific primer 12 GCGGATCCGA CATCAAGGGC AGCG 24 44base pairs nucleic acid single linear DNA (genomic) Primer 3 13CGCGGATCCA TATGGACGCA TCATTACTCC TCTCCGTCGC GCTC 44 31 base pairsnucleic acid single linear DNA (genomic) Primer 4 14 CGCAAGCTTATTACATCTCA ACGGGGACCC T 31 41 base pairs nucleic acid single linear DNA(genomic) Primer 5 15 CGCGGATCCA TATGGCAACA ACAGCAACCC CGCAGCTCCT C 4139 base pairs nucleic acid single linear DNA (genomic) Primer 6 16CGCAAGCTTA TTATGCTGCG CGGCGGTTCT TGTATTTGG 39 542 amino acids amino acidunknown linear protein NO NO internal Sinapis alba 17 Met Asn Thr PheThr Ser Asn Ser Ser Asp Leu Thr Ser Thr Thr Lys 1 5 10 15 Gln Thr LeuSer Phe Ser Asn Met Tyr Leu Leu Thr Thr Leu Gln Ala 20 25 30 Phe Val AlaIle Thr Leu Val Met Leu Leu Lys Lys Val Leu Val Asn 35 40 45 Asp Thr AsnLys Lys Lys Leu Ser Leu Pro Pro Gly Pro Thr Gly Trp 50 55 60 Pro Ile IleGly Met Val Pro Thr Met Leu Lys Ser Arg Pro Val Phe 65 70 75 80 Arg TrpLeu His Ser Ile Met Lys Gln Leu Asn Thr Glu Ile Ala Cys 85 90 95 Val ArgLeu Gly Ser Thr His Val Ile Thr Val Thr Cys Pro Lys Ile 100 105 110 AlaArg Glu Val Leu Lys Gln Gln Asp Ala Leu Phe Ala Ser Arg Pro 115 120 125Met Thr Tyr Ala Gln Asn Val Leu Ser Asn Gly Tyr Lys Thr Cys Val 130 135140 Ile Thr Pro Phe Gly Glu Gln Phe Lys Lys Met Arg Lys Val Val Met 145150 155 160 Thr Glu Leu Val Cys Pro Ala Arg His Arg Trp Leu His Gln LysArg 165 170 175 Ala Glu Glu Asn Asp His Leu Thr Ala Trp Val Tyr Asn MetVal Asn 180 185 190 Asn Ser Asp Ser Val Asp Phe Arg Phe Val Thr Arg HisTyr Cys Gly 195 200 205 Asn Ala Ile Lys Lys Leu Met Phe Gly Thr Arg ThrPhe Ser Gln Asn 210 215 220 Thr Ala Pro Asn Gly Gly Pro Thr Ala Glu AspIle Glu His Met Gly 225 230 235 240 Ala Met Phe Glu Ala Leu Gly Phe ThrPhe Ser Phe Cys Ile Ser Asp 245 250 255 Tyr Leu Pro Ile Leu Thr Gly LeuAsp Leu Asn Gly His Glu Lys Ile 260 265 270 Met Arg Asp Ser Ser Ala IleMet Asp Lys Tyr His Asp Pro Ile Ile 275 280 285 Asp Ala Arg Ile Lys MetTrp Arg Glu Gly Lys Lys Thr Gln Ile Glu 290 295 300 Asp Phe Leu Asp IlePhe Ile Ser Ile Lys Asp Glu Glu Gly Asn Pro 305 310 315 320 Leu Leu ThrAla Asp Glu Ile Lys Pro Thr Ile Lys Glu Leu Val Met 325 330 335 Ala AlaPro Asp Asn Pro Ser Asn Ala Val Glu Trp Ala Met Ala Glu 340 345 350 MetVal Asn Lys Pro Glu Ile Leu Arg Lys Ala Met Glu Glu Ile Asp 355 360 365Arg Val Val Gly Lys Glu Arg Leu Val Gln Glu Ser Asp Ile Pro Lys 370 375380 Leu Asn Tyr Val Lys Ala Ile Leu Arg Glu Ala Phe Arg Leu His Pro 385390 395 400 Val Ala Ala Phe Asn Leu Pro His Val Ala Leu Ser Asp Ala ThrVal 405 410 415 Ala Gly Tyr His Ile Pro Lys Gly Ser Gln Val Leu Leu SerArg Tyr 420 425 430 Gly Leu Gly Arg Asn Pro Lys Val Trp Ala Asp Pro LeuSer Phe Lys 435 440 445 Pro Glu Arg His Leu Asn Glu Cys Ser Glu Val ThrLeu Thr Glu Asn 450 455 460 Asp Leu Arg Phe Ile Ser Phe Ser Thr Gly XaaArg Gly Cys Ala Ala 465 470 475 480 Pro Ala Leu Gly Thr Ala Leu Thr ThrMet Leu Leu Ala Arg Leu Leu 485 490 495 Gln Gly Phe Thr Trp Lys Leu ProGlu Asn Glu Thr Arg Val Glu Leu 500 505 510 Met Glu Ser Ser His Asp MetPhe Leu Ala Lys Pro Leu Val Met Val 515 520 525 Gly Glu Leu Arg Leu ProGlu His Leu Tyr Pro Thr Val Lys 530 535 540 6 amino acids amino acidsingle linear peptide NO NO internal 18 Lys Pro Glu Arg His Leu 1 5 6amino acids amino acid single linear peptide NO NO internal 19 Thr GlyLys Arg Gly Cys 1 5 31 amino acids amino acid single linear peptide NONO internal 20 Lys Pro Glu Arg His Leu Asn Glu Cys Ser Glu Val Thr LeuThr Glu 1 5 10 15 Asn Asp Leu Arg Phe Ile Ser Phe Ser Thr Gly Lys ArgGly Cys 20 25 30 31 amino acids amino acid single linear peptide NO NOinternal 21 Lys Pro Glu Arg His Phe Asn Glu Cys Ser Glu Val Thr Leu ThrGlu 1 5 10 15 Asn Asp Leu Arg Phe Ile Ser Phe Ser Thr Gly Lys Arg GlyCys 20 25 30 14 amino acids amino acid single linear peptide NO NON-terminal 22 Gly Leu Val Lys Glu Gly Val Asp Met Glu Glu Gly Thr Leu 15 10 15 amino acids amino acid single linear peptide NO NO N-terminal 23Met Gly Leu Val Lys Glu Gly Val Asp Met Glu Glu Gly Thr Leu 1 5 10 15

What is claimed is:
 1. A DNA molecule coding for a cytochrome P450 monooxygenase catalyzing the conversion of an aldoxime to a nitrile and the conversion of said nitrile to the corresponding cyanohydrin.
 2. The DNA molecule according to claim 1, wherein the monooxygenase is obtainable from plants which produce cyanogenic glycosides.
 3. The DNA molecule according to claim 2, wherein the monooxygenase is obtainable from plants selected from the group consisting of the genera Sorghum, Trifolium, Linum, Taxus, Triglochin, Mannihot, Amygdalus, Prunus and cruciferous plants.
 4. The DNA molecule according to claim 1, wherein the aldoxime is the result of the conversion of an amino acid selected from the group consisting of tyrosine, phenylalanine, tryptophan, valine, leucine, isoleucine and cyclopentenylglycine in the presence of a monooxygenase catalyzing the conversion of said amino acids to the corresponding N-hydroxyamino acids and the conversion of said N-hydroxyamino acids to said aldoximes.
 5. The DNA molecule according to claim 1, wherein the monooxygenase catalyzes more than one reaction of the biosynthetic pathway of cyanogenic glycosides.
 6. The DNA molecule according to claim 1 comprising DNA necessary for the use in recombinant DNA technology.
 7. The DNA molecule according to claim 1 coding for a monooxygenase of Sorghum bicolor.
 8. A cytochrome P450 monooxygenase which catalyzes the conversion of an aldoxime to a nitrile and the conversion of said nitrile to the corresponding cyanohydrine.
 9. The cytochrome P450 monooxygenase according to claim 8, whose ability to convert an aldoxime to a nitrile depends on the presence of NADPH and which dependency can be overcome by the addition of reductants.
 10. The cytochrome P450 monooxygenase according to claim 9 having a molecular weight of 55 kD as determined by SDS-PAGE and an N-terminal sequence as described in SEQ ID NO:
 3. 11. A method for the isolation of a cDNA molecule coding for a cytochrome P450 monooxygenase, which monooxygenase catalyzes the conversion of an aldoxime to a nitrile and the conversion of said nitrile to the corresponding cyanohydrin; comprising (a) isolating and solubilizing microsomes from plant tissue producing cyanogenic glycosides, (b) purifying the cytochrome P450 monooxygenase, (c) raising antibodies against the purified monooxygenase, (d) probing a cDNA expression library of plant tissue producing cyanogenic glycosides with said antibody, and (e) isolating clones which express the monooxygenase.
 12. A method for the isolation of a cDNA molecule coding for a cytochrome P450 monooxygenase which catalyzes the conversion of an aldoxime to a nitrile and the conversion of said nitrile to the corresponding cyanohydrin; comprising (a) isolating and solubilizing microsomes from plant tissue producing cyanogenic glycosides, (b) purifying the cytochrome P450 monooxygenase, (c) obtaining a complete or partial protein sequence of the monoxygenase, (d) designing oligonucleotides specifying DNA coding for 4 to 15 amino acids of said monooxygenase protein sequence (e) probing a cDNA library of plant tissue producing cyanogenic glycosides with said oligonucleotides, or DNA molecules obtained from PCR amplification of cDNA using said oligonucleotides, and (f) isolating clones which encode cytochrome P450 monooxygenase.
 13. A method for the isolation of a cDNA molecule coding for a cytochrome P450 monooxygenase which catalyzes the conversion of an aldoxime to a nitrite and the conversion of said nitrile to the corresponding cyanohydrin; comprising (a) designing degenerated oligonucleotides covering 3 to 10 amino acids of conserved regions of A-type cytochromes P450, (b) using the degenerated oligonucleotides to amplify one or more cytochrome specific DNA fragments using the polymerase chain reaction, (c) screening a cDNA library with the cytochrome specific fragments to obtain full length cDNA, (d) expressing the full length cDNA in a microbial host, (e) identifying hosts expressing cytochrome P450 monooxygenase which catalyzes the conversion of an aldoxime to a nitrile and the conversion of said nitrite to the corresponding cyanohydrin, and (f) purifying the cloned DNA from said host.
 14. A method for producing a purified recombinant cytochrome P450 monooxygenase which catalyzes the conversion of an aldoxime to a nitrile and the conversion of said nitrite to the corresponding cyanohydrine; comprising (a) engineering the gene encoding said monooxygenase to be expressible in a host organism, (b) transforming said host organism with the engineered gene, and (c) isolating the protein from the host organism or its culture supernatant.
 15. The method according to claim 14, wherein the host organism is selected from the group consisting of bacteria, yeast and insect cells.
 16. The method according to claim 14, wherein the cytochrome P450 monooxygenase of Sorghum bicolor is produced.
 17. The method according to claim 14, wherein the cytochrome P450 monooxygenase has been modified.
 18. A transgenic plant comprising stably integrated into its genome DNA coding for a monooxygenase according to claim 8 or DNA according to claim 1 encoding sense RNA, anti sense RNA or a ribozyme, the expression of which reduces expression of cytochrome P450 monooxygenase.
 19. The transgenic plant according to claim 18 selected from the group consisting of plant types consisting of Cereals, Protein Crops, Fruit Crops, Vegetables and Tubers, Nuts, Oil Crops, Sugar Crops, Forage and Turf Grasses, Forage Legumes, Fiber Plants and Woody Plants, Drug Crops and Spices and Flavorings.
 20. The transgenic maize plant according to claim
 18. 21. The transgenic barley plant according to claim
 18. 22. A method for obtaining a transgenic plant according to claim 18 comprising (a) introducing into a plant cell or tissue which can be regenerated to a complete plant, DNA comprising a gene expressible in that plant encoding a monooxygenase according to claim 8, and (b) selecting transgenic plants.
 23. A method for obtaining a transgenic plant according to claim 18 comprising (a) introducing into a plant cell or tissue which can be regenerated to a complete plant, DNA encoding sense RNA, anti sense RNA or a ribozyme, the expression of which reduces the expression of cytochrome P450 monooxygenases according to claim 1, and (b) selecting transgenic plants.
 24. Use of a DNA molecule according to claim 1 to obtain transgenic plants according to claim
 18. 25. A method of using a DNA molecule according to claim 1 to obtain a transgenic plant resistant to insects, acarids, or nematodes comprising (a) introducing into a plant cell or tissue which can be regenerated to a complete plant, DNA comprising a gene expressible in that plant encoding a monooxygenase according to claims 8, (b) selecting transgenic plants, and (c) identifying plants which are resistant to insects, acarids, or nematodes.
 26. The method according to claim 22, wherein said plant is a monocot or dicot plant selected from the group of plant types consisting of Cereals, Protein Crops, Fruit Crops, Vegetables and Tubers, Nuts, Oil Crops, Sugar Crops, Forage and Turf Grasses, Forage Legumes, Fiber Plants and Woody Plants, Drug Crops and Spices and Flavorings.
 27. A cytochrome P450 enzyme catalyzing the first step in the biosynthesis of glucosinolates.
 28. The enzyme of claim 27 having the amino acid sequence shown in SEQ ID NO:17. 